Compounds that participate in cooperative binding and uses thereof

ABSTRACT

The invention features compounds (e.g., macrocyclic compounds) capable of modulating biological processes, for ex ample through binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein such as CEP250. These compounds bind endogenous intracellular presenter proteins, such as the FKBPs or cyclophilins, and the resulting binary complexes selectively bind and modulate the activity of the target protein. Formation of a tripartite complex among the presenter protein, the compound, and the target protein is driven by both protein-compound and protein-protein interactions, and both are required for modulation of target protein activity.

BACKGROUND

CEP250 is a core centrosomal protein localized to the proximal ends ofcentrioles where it contributes to centrosome-centrosome cohesion duringinterphase of the cell cycle. Centrioles are essential for the formationof centrosomes and cilia (e.g., motile cilia or non-motile cilia). Thus,the compounds that modulate CEP250 may be useful in the bindingstabilization, or modulation of the activity of one or more componentsof the centrosome or cilia. These compounds may also be used to modulatesignal transduction pathways associated with CEP250, including, but notlimited to, Hedgehog, Wnt, PDGFRalpha, and integrin signaling, and thetreatment of diseases or disorders related to centrosome aberrations(e.g., cancer or ciliopathies) or Hedgehog, Wnt, PDGFRalpha, or integrinsignaling.

The present invention is related to compounds that modulate the activityof of target proteins such as CEP250 and, therefore, may be useful inthe treatment of diseases and disorders such as cancer, ciliopathies, orinfections.

SUMMARY

The invention features compounds (e.g., macrocyclic compounds) capableof modulating the activity of target proteins such as CEP250 throughinteraction with presenter proteins (e.g., FKBP12, FKBP12.6, FKBP25,FKBP52, cyclophilin A) and the target protein. These compounds may beuseful in the treatment of diseases and disorders such as cancer,ciliopathies, or infections.

In one aspect, the invention features a compound (e.g., a macrocycliccompound comprising 14 to 40 ring atoms). The compound includes: (a) atarget protein interacting moiety (e.g., a CEP250 interacting moiety);and (b) a presenter protein binding moiety; wherein the compound and apresenter protein form a complex that specifically binds to the targetprotein. In some embodiments, each of the compound and the presenterprotein do not substantially bind to the target protein in the absenceof forming the complex; or the compound and a presenter protein form acomplex that binds to the target protein with at least 5-fold greateraffinity than the affinity of each of the compound and the presenterprotein to the target protein in the absence of forming said complex; ora pharmaceutically acceptable salt thereof.

In some embodiments, a provided compound includes one or more linkermoieties. In some embodiments, a linker moiety connects the presenterprotein binding moiety (or portion thereof) and the target proteininteracting moiety (or portion thereof).

In some embodiments, the compound has the structure:

wherein A includes a target protein interacting moiety (e.g., a CEP250interacting moiety);

B includes a presenter protein binding moiety; and

L¹ and L² are independently selected from a bond and a linear chain ofup to 10 atoms, independently selected from carbon, nitrogen, oxygen,sulfur or phosphorous atoms, wherein each atom in the chain isoptionally substituted with one or more substituents independentlyselected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo,bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino,dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio,arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, andsulfonyl, and wherein any two atoms in the chain may be taken togetherwith the substituents bound thereto to form a ring, wherein the ring maybe further substituted and/or fused to one or more optionallysubstituted carbocyclic, heterocyclic, aryl, or heteroaryl rings.

In some embodiments, the compound has the structure:

wherein each of Z¹ and Z² are, independently, hydrogen or hydroxyl.

In some embodiments, at least one atom of L¹, L², Z¹, and/or Z²participates in binding to the presenter protein and the target protein.In certain embodiments, at least one atom of L¹, L², Z¹, and/or Z² doesnot participate in binding to the presenter protein or the targetprotein.

In some embodiments, L¹, L², Z¹, and/or Z² includes one or more atomsthat participates in binding to the presenter protein and/or to thetarget protein. In some embodiments, at least one atom of one or more ofL¹, L², Z¹, and/or Z² participates in binding to the presenter proteinand/or the target protein. In certain embodiments, at least one atom ofone or more of L¹, L², Z¹, and/or Z² does not participate in binding tothe presenter protein and/or the target protein.

In some embodiments, the presenter protein binding moiety includes 5 to20 ring atoms (e.g., 5 to 10, 7 to 12, 10 to 15, 12 to 17, or 15 to 20ring atoms).

In some embodiments, the compound consists of 14 to 20 ring atoms (e.g.,14 to 16, 14 to 17, 15 to 18, 16 to 19, or 17 to 20 ring atoms or 14,15, 16, 17, 18, 19, or 20 ring atoms). In certain embodiments, thecompound consists of 21 to 26 ring atoms (e.g., 21 to 23, 22 to 24, 23to 25, or 24 to 26 ring atoms or 21, 22, 23, 24, 25, 26 ring atoms). Insome embodiments, the compound consists of 27 to 40 ring atoms (e.g., 27to 30, 29 to 34, 33 to 38, 37 to 40 ring atoms or 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 ring atoms).

In some embodiments, the presenter protein binding moiety includes thestructure of Formula I:

wherein n is 0 or 1;

X¹ and X³ are each independently O, S, CR³R⁴, or NR⁵;

X² is O, S, or NR⁵;

R¹, R², R³, and R⁴ are each independently hydrogen, hydroxyl, optionallysubstituted amino, halogen, thiol, optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted C₁-C₆ heteroalkyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆heteroalkynyl, optionally substituted C₃-C₁₀ carbocyclyl, optionallysubstituted C₆-C₁₀ aryl, optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heteroaryl, optionally substituted C₂-C₉heteroaryl C₁-C₆ alkyl, optionally substituted C₂-C₉ heterocyclyl, oroptionally substituted C₂-C₉ heterocyclyl C₁-C₆ alkyl, or any two of R¹R², R³, or R⁴ are taken together with the atom or atoms to which theyare bound to form an optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl; and

each R⁵ is, independently, hydrogen, hydroxyl, optionally substitutedC₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, optionallysubstituted C₂-C₆ alkynyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆heteroalkynyl, optionally substituted C₃-C₁₀ carbocyclyl, optionallysubstituted C₆-C₁₀ aryl, optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heteroaryl, optionally substituted C₂-C₉heteroaryl C₁-C₆ alkyl, optionally substituted C₂-C₉ heterocyclyl, oroptionally substituted C₂-C₉ heterocyclyl C₁-C₆ alkyl, or R⁵ and one ofR¹, R², R³, or R⁴ are taken together with the atom or atoms to whichthey are bound to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl.

In some embodiments, X¹ is connected to L¹ and X³ is connected to L². Insome embodiments, X¹ is connected to L² and X³ is connected to L¹.

In some embodiments, the presenter protein binding moiety includes thestructure of Formula Ia:

In some embodiments, the presenter protein binding moiety is or includesthe structure of any one of Formulae II-IV:

wherein o, and p are independently 0 or 1;

q is an integer between 0 and 7;

r is an integer between 0 and 4;

X⁴ and X⁵ are each, independently, CH₂, O, S, SO, SO₂, or NR¹³;

each R⁶ and R⁷ are independently hydrogen, hydroxyl, optionallysubstituted amino, halogen, thiol, optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted C₁-C₆ heteroalkyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆heteroalkynyl, optionally substituted C₃-C₁₀ carbocyclyl, optionallysubstituted C₆-C₁₀ aryl, optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heteroaryl, optionally substituted C₂-C₉heteroaryl C₁-C₆ alkyl, optionally substituted C₂-C₉ heterocyclyl,optionally substituted C₂-C₉ heterocyclyl C₁-C₆ alkyl, or R⁶ and R⁷combine with the carbon atom to which they are bound to form C═O or R⁶and R⁷ combine to form an optionally substituted C₃-C₁₀ carbocyclyl oroptionally substituted C₂-C₉ heterocyclyl;

each R⁸ is, independently, hydroxyl, optionally substituted amino,halogen, thiol, optionally substituted C₁-C₆ alkyl, optionallysubstituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ heteroalkynyl, optionallysubstituted C₃-C₁₀ carbocyclyl, optionally substituted C₆-C₁₀ aryl,optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl, optionally substitutedC₂-C₉ heteroaryl, optionally substituted C₂-C₉ heteroaryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heterocyclyl, or optionally substitutedC₂-C₉ heterocyclyl C₁-C₆ alkyl or two R⁸ combine to form an optionallysubstituted C₃-C₁₀ carbocyclyl, optionally substituted C₆-C₁₀ aryl, oroptionally substituted C₂-C₉ heteroaryl;

R⁹ is optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substitutedC₁-C₆ heteroalkyl, optionally substituted C₂-C₆ heteroalkenyl,optionally substituted C₂-C₆ heteroalkynyl, optionally substitutedC₃-C₁₀ carbocyclyl, optionally substituted C₆-C₁₀ aryl, optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl, optionally substituted C₂-C₉heteroaryl, optionally substituted C₂-C₉ heteroaryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heterocyclyl, or optionally substitutedC₂-C₉ heterocyclyl C₁-C₆ alkyl;

R¹⁰ is optionally substituted C₁-C₆ alkyl;

each R¹¹ is, independently, hydroxyl, cyano, optionally substitutedamino, halogen, thiol, optionally substituted C₁-C₆ alkyl, optionallysubstituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ heteroalkynyl, optionallysubstituted C₃-C₁₀ carbocyclyl, optionally substituted C₆-C₁₀ aryl,optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl, optionally substitutedC₂-C₉ heteroaryl, optionally substituted C₂-C₉ heteroaryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heterocyclyl, or optionally substitutedC₂-C₉ heterocyclyl C₁-C₆ alkyl or two R¹¹ combine to form an optionallysubstituted C₃-C₁₀ carbocyclyl, optionally substituted C₆-C₁₀ aryl, oroptionally substituted C₂-C₉ heteroaryl; and

R¹² and R¹³ are each, independently, optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted aryl, C₃-C₇ carbocyclyl, optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl, and optionally substitutedC₃-C₇carbocyclyl C₁-C₆ alkyl.

In some embodiments, L¹ is connected to the left of the presenterprotein binding moiety and L² is connected to the right of the presenterprotein binding moiety. In some embodiments, L¹ is connected to theright of the presenter protein binding moiety and L² is connected to theleft of the presenter protein binding moiety.

In some embodiments, the presenter protein binding moiety is or includesthe structure of any one of Formulae IIa-IVa:

In some embodiments, the presenter protein binding moiety is or includesthe structure of Formula V:

wherein R¹⁴ is hydrogen, hydroxyl, optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted C₁-C₆ heteroalkyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆heteroalkynyl, optionally substituted C₃-C₁₀ carbocyclyl, optionallysubstituted C₆-C₁₀ aryl, optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heteroaryl, optionally substituted C₂-C₉heteroaryl C₁-C₆ alkyl, optionally substituted C₂-C₉ heterocyclyl,optionally substituted C₂-C₉ heterocyclyl C₁-C₆ alkyl.

In certain embodiments, the presenter protein binding moiety is orincludes the structure of Formula VI or VII:

wherein s and t are each, independently, an integer from 0 to 7;

X⁶ and X⁷ are each, independently, O, S, SO, SO₂, or NR¹⁹;

R¹⁵ and R¹⁷ are each, independently, hydrogen hydroxyl, or optionallysubstituted C₁-C₆ alkyl;

R¹⁶ and R¹⁸ are each, independently, hydroxyl, optionally substitutedamino, halogen, thiol, optionally substituted C₁-C₆ alkyl, optionallysubstituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ heteroalkynyl, optionallysubstituted C₃-C₁₀ carbocyclyl, optionally substituted C₆-C₁₀ aryl,optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl, optionally substitutedC₂-C₉ heteroaryl, optionally substituted C₂-C₉ heteroaryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heterocyclyl, or optionally substitutedC₂-C₉ heterocyclyl C₁-C₆ alkyl; and

R¹⁹ is optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substitutedaryl, C₃-C₇ carbocyclyl, optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl,and optionally substituted C₃-C₇ carbocyclyl C₁-C₆ alkyl.

In certain embodiments, the presenter protein binding moiety is orincludes the structure:

In certain embodiments, the presenter protein binding moiety is orincludes the structure:

In some embodiments, the presenter protein binding moiety is or includesthe structure:

In some embodiments, presenter protein binding moiety is or includes thestructure:

In certain embodiments, the presenter protein binding moiety has thestructure:

In certain embodiments, the target protein interacting moiety (e.g.,CEP250 interacting moiety) is or includes the structure of Formula XIII:

where the dotted lines represent zero to three double bonds, providedthat no two double bonds are adjacent to one another;

R³¹ and R³² are independently hydrogen, hydroxyl, optionally substitutedamino, halogen, thiol, optionally substituted amino acid, optionallysubstituted C₁-C₆ acyl, optionally substituted C₁-C₆ alkyl, optionallysubstituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ heteroalkynyl, optionallysubstituted C₃-C₁₀ cycloalkyl, optionally substituted C₄-C₁₀cycloalkenyl, optionally substituted C₄-C₁₀ cycloalkynyl, optionallysubstituted C₆-C₁₀ aryl, optionally substituted C₆-C₁₀ aryl C₁-C₆ alkyl,optionally substituted C₂-C₉ heteroaryl, optionally substituted C₂-C₉heteroaryl C₁-C₆ alkyl, optionally substituted C₂-C₉ heterocyclyl,optionally substituted C₂-C₉ heterocyclyl C₁-C₆ alkyl, or R³¹ and R³²combine to form C═O; and

R³³ is hydrogen or C═O, provided that no double bonds are adjacent to aC═O group.

In certain embodiments, the target protein interacting moiety (e.g.,CEP250 interacting moiety) is or includes the structure of FormulaXIIIa:

In some embodiments, the target protein interacting moiety (e.g., CEP250interacting moiety) is or includes the structure:

In some embodiments, the target protein interacting moiety (e.g., CEP250interacting moiety) is or includes the structure:

In certain embodiment, R³¹ is hydrogen. In some embodiments, R³² ishydroxyl.

In certain embodiments, the target protein interacting moiety (e.g.,CEP250 interacting moiety) is or includes the structure:

In certain embodiments, the target protein interacting moiety (e.g.,CEP250 interacting moiety) does not have the structure:

In some embodiments, the compound has the structure of Formula XV:

wherein u is 0 or 1.

In certain embodiments, X⁴ is CH₂. In some embodiments, X⁵ is O. Incertain embodiments, X⁷ is O. In some embodiments, t is 1. In certainembodiments, R¹⁸ is optionally substituted C₁-C₆ alkyl (e.g., methyl).In some embodiments, R¹⁴ is optionally substituted C₆-C₁₀ aryl C₁-C₆alkyl.

In certain embodiments, the compound has the structure:

wherein v is 1 or 2;

Ar is optionally substituted aryl or optionally substituted heteroaryl;and

each R³⁴ and each R³⁵ is independently hydrogen, hydroxyl, optionallysubstituted amino, halogen, thiol, optionally substituted amino acid,optionally substituted C₁-C₆ acyl, optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted C₁-C₆ heteroalkyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆heteroalkynyl, optionally substituted C₃-C₁₀ cycloalkyl, optionallysubstituted C₄-C₁₀ cycloalkenyl, optionally substituted C₄-C₁₀cycloalkynyl, optionally substituted C₆-C₁₀ aryl, optionally substitutedC₆-C₁₀ aryl C₁-C₆ alkyl, optionally substituted C₂-C₉ heteroaryl,optionally substituted C₂-C₉ heteroaryl C₁-C₆ alkyl, optionallysubstituted C₂-C₉ heterocyclyl, or optionally substituted C₂-C₉heterocyclyl C₁-C₆ alkyl.

In some embodiments, L¹ and L² are both a single bond.

In certain embodiments, the compound has the structure:

In some embodiments, u is 0 and o is 1. In some embodiments, u is 1 ando is 0. In certain embodiments, v is 2. In some embodiments, Ar isoptionally substituted aryl (e.g., phenyl or 3-hydroxy-phenyl). In someembodiments, R³⁵ is hydrogen. In certain embodiments, at least one R³⁴is optionally substituted C₁-C₆ alkyl (e.g., ethyl). In someembodiments, R³¹ is hydroxyl. In some embodiments, R³² is hydrogen. Incertain embodiments, q is 0. In some embodiments, R¹⁷ is hydroxyl.

In some embodiments, u is 0, p is 1, and o is 1. In certain embodiments,R⁶ and R⁷ are both hydrogen. In some embodiments, u is 1, p is 1, and ois 0. In some embodiments, R⁶ and R⁷ combine to form C═O. In someembodiments, u is 1, p is 0, and o is 0.

In some embodiments, at least one Y is an N-alkylated amino acid (e.g.,an N-methyl amino acid). In some embodiments, at least one Y is aD-amino acid. In some embodiments, at least one Y is a non-natural aminoacid. In certain embodiments, at least one Y includes a depsi-linkage.

In some embodiments, the compound does not include the structure:

In some embodiments, the portion of the molecule that comprises eachring atom that participates in binding to the target protein has a c LogP greater than 2 (e.g., greater than 3, greater than 4, greater than 5,greater than 6). In certain embodiments, the portion of the moleculethat comprises each ring atom that participates in binding to the targetprotein has a polar surface area less than 350 Å² (e.g., less than 300Å², less than 250 Å², less than 200 Å², less than 150 Å², less than 125Å²). In some embodiments, the portion of the molecule that compriseseach ring atom that participates in binding to the target proteinincludes at least one atom of a linker.

In some embodiments, the compound has a molecular weight between 400 and2000 Daltons (e.g., 400 to 600, 500 to 700, 600 to 800, 700 to 900, 800to 1000, 900 to 1100, 1000 to 1200, 1100 to 1300, 1200 to 1400, 1300 to1500, 1400 to 1600, 1500 to 1700, 1600 to 1800, 1700 to 1900, or 1800 to2000 Daltons). In certain embodiments, the compound has an even numberof ring atoms (e.g., 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,or 40 ring atoms). In some embodiments, the compound is cell penetrant.In some embodiments, the compound is substantially pure (e.g., compoundis provided in a preparation that is substantially free of contaminantssuch as other compounds and/or components of a cell lysate. In certainembodiments, the compound is isolated. In some embodiments, the compoundis an engineered compound. In some embodiments, the compound isnon-naturally occurring.

In certain embodiments, the complex binds to the target protein with atleast 5-fold greater (e.g., at least 10-fold greater, at least 20-foldgreater, at least 50-fold greater, or at least 100-fold greater)affinity than the complex binds to mTOR and/or calcineurin. In someembodiments, the complex binds to the target protein with at least5-fold greater (e.g., at least 10-fold greater, at least 20-foldgreater, at least 50-fold greater, or at least 100-fold greater)affinity than the affinity of the compound to the target protein whenthe compound is not bound in a complex with the presenter protein. Insome embodiments, the complex binds to the target protein with at least5-fold greater (e.g., at least 10-fold greater, at least 20-foldgreater, at least 50-fold greater, or at least 100-fold greater)affinity than the affinity of the presenter protein to the targetprotein when the presenter protein is not bound in a complex with thecompound. In certain embodiments, the complex inhibits the naturallyoccurring interaction between the target protein and a ligand thatspecifically binds the target protein.

In some embodiments, the presenter protein is a prolyl isomerase (e.g.,a member of the FKBP family such as FKBP12, FKBP12.6, FKBP25, or FKBP52,a member of the cyclophilin family such as PP1A, CYPB, CYPC, CYP40,CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6,RANBP2, PPWD1, or PIN1).

In certain embodiments, the target protein is a eukaryotic targetprotein. In some embodiments, the eukaryotic target protein is a fungaltarget protein. In certain embodiments, the target protein is aprokaryotic target protein such as a bacterial target protein.

In some embodiments, the target protein is CEP250.

In an aspect, the invention features a compound, or a stereoisomer, orpharmaceutically acceptable salt thereof selected from any one ofcompounds 1-11 in Table 1.

TABLE 1 Found Molecular Chemical LC-MS Structure # Weight Formula cLogP[M + Na]+

 1 595.82 C₃₆H₅₃NO₆ 6.7 618.4

 2 609.80 C₃₆H₅₁NO₇ 6.3 632.4

 3 623.79 C₃₆H₄₉NO₈ 5.2 646.3

 4 595.78 C₃₅H₄₉NO₇ 5.9 618.3

 5 623.79 C₃₆H₄₉NO₈ 4.9 646.3

 6 613.82 C₃₆H₅₅NO₇ 636.4

 7 625.80 C₃₆H₅₁NO₈ 648.4

 8 625.80 C₃₆H₅₁NO₈ 648.4

 9 607.79 C36H49NO7 630.3

10 627.82 C36H53NO8 650.4

11 627.82 C36H53NO8 650.3

In other embodiments, the compound has the structure of Formula XXVII:

In certain embodiments, the compound has the structure of Formula XXII:

wherein R³⁶ is hydrogen, optionally substituted hydroxyl, or optionallysubstituted amino.

In some aspects, the invention features a presenter protein/compoundcomplex including any of the compounds of the invention and a presenterprotein.

In some embodiments of the presenter protein/compound complex, thepresenter protein is a protein encoded by any one of the genes or ahomolog thereof of Table 2. In some embodiments of the presenterprotein/compound complex the presenter protein is a prolyl isomerase(e.g., a member of the FKBP family such as FKBP12, FKBP12.6, FKBP25, orFKBP52, a member of the cyclophilin family such as PP1A, CYPB, CYPC,CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4,PPIL6, RANBP2, PPWD1, or PIN1).

In some aspects, the invention features a pharmaceutical compositionincluding any of the compounds or complexes of the invention and apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is in unit dosage form.

In some aspects, the invention features a method of modulating thetarget protein. In some embodiments, such a method includes steps ofcontacting the target protein with a modulating (e.g., positive ornegative modulation) amount of any of the compounds (e.g., in thepresence of a presenter protein), presenter protein/compound complexes,or compositions of the invention.

In some aspects, the invention features a method of modulating (e.g.,positively or negatively modulating) the target protein. In someembodiments, such a method includes steps of contacting a cellexpressing the target protein and a presenter protein with an effectiveamount of a compound or composition of the invention under conditionswherein the compound can form a complex with the presenter protein andthe resulting complex can bind to the target protein, thereby modulating(e.g., positively or negatively modulating) the target protein.

In some aspects, the invention features a method of modulating (e.g.,positively or negatively modulating) the target protein. In someembodiments, such a method includes steps of contacting the targetprotein with a presenter protein/compound complex of the invention,thereby modulating the target protein.

In some aspects, the invention features a method of inhibiting prolylisomerase activity. In some embodiments, such a method includescontacting a cell expressing the prolyl isomerase with a compound orcomposition of the invention under conditions that permit the formationof a complex between the compound and the prolyl isomerase, therebyinhibiting the prolyl isomerase activity.

In some aspects, the invention features a method of forming a presenterprotein/compound complex in a cell. In some embodiments, such a methodincludes steps of contacting a cell expressing the presenter proteinwith a compound or composition of the invention under conditions thatpermit the formation of a complex between the compound and the presenterprotein.

In some aspects, the invention features a method of treating cancer,such as medullablastoma, basal cell carcinoma, lung cancer, pancreaticcancer, prostate cancer or glioma. This method includes administering aneffective amount of a compound, complex, or composition of the inventionto a subject in need thereof.

In some aspects, the invention features a method of treating cancer.This method includes contacting a cancer cell with an effective amountof a compound, complex, or composition of the invention.

In some aspects, the invention features method of treating cancer. Thismethod includes modulating CEP250 in a subject in need thereof bycontacting CEP250 with a modulating amount of a CEP250-binding compound,complex, or composition of the invention.

In some aspects, the invention features a method of treating cancer.This method includes forming any of the foregoing presenter proteincomplexes in a cell by contacting said cell with an effective amount ofany of the foregoing compounds or compositions.

In some aspects, the invention features a method of treating aciliopathy. This method includes administering an effective amount of acompound, complex, or composition of the invention to a subject in needthereof.

In some aspects, the invention features a method of treating aciliopathy. This method includes contacting a cell with an effectiveamount of a compound, complex, or composition of the invention.

In some aspects, the invention features a method of treating aciliopathy. The method includes modulating CEP250 in a subject in needtherof by contacting CEP250 with a modulating amount of a compound,complex, or composition of the invention.

In some aspects, the invention features a method of treating aciliopathy. This method includes forming any of the foregoing presenterprotein complexes in a cell by contacting said cell with an effectiveamount of any of the foregoing compounds or compositions.

In some aspects, the invention features a method of treating aninfection (e.g., a bacterial infection, fungal infection, or protozoalinfection). This method includes administering an effective amount of acompound, complex, or composition of the invention to a subject in needthereof.

In some aspects, the invention features a method of treating aninfection (e.g., a bacterial infection, fungal infection, or protozoalinfection). This method includes contacting a cell with an effectiveamount of a compound, complex, or composition of the invention.

In some aspects, the invention features a method of treating aninfection (e.g., a bacterial infection, fungal infection, or protozoalinfection). This method includes modulating CEP250 in a subject in needtherof by contacting CEP250 with a modulating amount of a CEP250-bindingcompound, complex, or composition of the invention.

In some aspects, the invention features a method of treating aninfection. This method includes forming any of the foregoing presenterprotein complexes in a cell by contacting said cell with an effectiveamount of any of the foregoing compounds or compositions.

In some aspects, the invention features a method for the preparation ofa compound of the invention. In some embodiments, such a method includessteps of culturing a bacterial strain of the genus Streptomyces andisolating the compound from the fermentation broth. In some embodiments,the bacterial strain is Streptomyces malaysiensis (NRRL B-24313; ATCCBAA-13; DSM 41697; JCM 10672; KCTC 9934; NBRC 16446; CGMCC 4.1900; IFO16448) or a natural variant thereof. In some embodiments, the bacterialstrain is an engineered strain. In some embodiments, the bacterialstrain is engineered in that it has been modified to produce thecompound and/or to secrete the compound into the broth.

In some embodiments, the present disclosure provides methods forpreparing a compound as described herein, the method comprising steps ofculturing a bacterial strain of the genus Streptomyces under conditionsin which the strain produces the compound and releases it into the andisolating the compound from the fermentation broth.

In some embodiments, a provided method comprises isolating a compound asdescribed herein from fermentation broth.

In some aspects, the invention features a tripartite complex including(i) a target protein and (ii) a presenter protein/compound complex, thepresenter protein/compound complex including a presenter protein and anyof the compounds of the invention.

In some embodiments of the tripartite complex, the presenterprotein/compound complex binds at a flat surface site on the targetprotein. In certain embodiments of the tripartite complex, the compound(e.g., macrocyclic compound) in the presenter protein/compound complexbinds at a hydrophobic surface site (e.g., a hydrophobic surface site onthe target protein including at least 30%, such as at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95%, hydrophobic residues) on the target protein. In someembodiments of the tripartite complex, the presenter protein/compoundcomplex binds to the target protein at a site of a naturally occurringprotein-protein interaction between the target protein and a proteinthat specifically binds the target protein. In some embodiments of thetripartite complex, presenter protein/compound complex does not bind atan active site of the target protein. In certain embodiments of thetripartite complex, presenter protein/compound complex binds at anactive site of the target protein.

In some embodiments of the tripartite complex, the structuralorganization of the compound (e.g., macrocyclic compound) issubstantially unchanged in the tripartite complex compared to thecompound (e.g., macrocyclic compound) in the presenter protein/compoundcomplex but not in the tripartite complex.

In certain embodiments of the tripartite complex, at least 10% (e.g., atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90%) of the total buried surfacearea of the target protein in the tripartite complex includes one ormore atoms that participate in binding to the compound (e.g.,macrocyclic compound). In some embodiments of the tripartite complex, atleast 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at or least 90%) of the totalburied surface area of the target protein in the tripartite complexincludes one or more atoms that participate in binding to the presenterprotein.

In some embodiments of the tripartite complex, the compound (e.g.,macrocyclic compound) contributes at least 10% (e.g., at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%) of the total binding free energy of thetripartite complex. In certain embodiments of the tripartite complex,the presenter protein contributes at least 10% (e.g., at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%) of the total binding free energy of thetripartite complex.

In some embodiments of the tripartite complex, at least 70% (e.g., atleast 80%, at least 90%, or at least 95%) of binding interactionsbetween one or more atoms of the compound (e.g., macrocyclic compound)and one or more atoms of the target protein are van der Waalsinteractions and/or T_(r)-effect interactions.

In some aspects, the invention features a compound collection comprisinga plurality of compounds (e.g., of macrocyclic compounds as describedherein). In some embodiments, compound collections include a pluralityof compounds that are variants of one another.

Chemical Terms

Those skilled in the art will appreciate that certain compoundsdescribed herein can exist in one or more different isomeric (e.g.,stereoisomers, geometric isomers, tautomers) and/or isotopic (e.g., inwhich one or more atoms has been substituted with a different isotope ofthe atom, such as hydrogen substituted for deuterium) forms. Unlessotherwise indicated or clear from context, a depicted structure can beunderstood to represent any such isomeric or isotopic form, individuallyor in combination.

Compounds described herein can be asymmetric (e.g., having one or morestereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

In some embodiments, one or more compounds depicted herein may exist indifferent tautomeric forms. As will be clear from context, unlessexplicitly excluded, references to such compounds encompass all suchtautomeric forms. In some embodiments, tautomeric forms result from theswapping of a single bond with an adjacent double bond and theconcomitant migration of a proton. In certain embodiments, a tautomericform may be a prototropic tautomer, which is an isomeric protonationstates having the same empirical formula and total charge as a referenceform. Examples of moieties with prototropic tautomeric forms areketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs,amide-imidic acid pairs, enamine-imine pairs, and annular forms where aproton can occupy two or more positions of a heterocyclic system, suchas, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole, and 1H- and 2H-pyrazole. In some embodiments, tautomericforms can be in equilibrium or sterically locked into one form byappropriate substitution. In certain embodiments, tautomeric formsresult from acetal interconversion, e.g., the interconversionillustrated in the scheme below:

Those skilled in the art will appreciate that, in some embodiments,isotopes of compounds described herein may be prepared and/or utilizedin accordance with the present invention. “Isotopes” refers to atomshaving the same atomic number but different mass numbers resulting froma different number of neutrons in the nuclei. For example, isotopes ofhydrogen include tritium and deuterium. In some embodiments, an isotopicsubstitution (e.g., substitution of hydrogen with deuterium) may alterthe physiciochemical properties of the molecules, such as metabolismand/or the rate of racemization of a chiral center.

As is known in the art, many chemical entities (in particular manyorganic molecules and/or many small molecules) can adopt a variety ofdifferent solid forms such as, for example, amorphous forms and/orcrystalline forms (e.g., polymorphs, hydrates, solvates, etc). In someembodiments, such entities may be utilized in any form, including in anysolid form. In some embodiments, such entities are utilized in aparticular form, for example in a particular solid form.

In some embodiments, compounds described and/or depicted herein may beprovided and/or utilized in salt form.

In certain embodiments, compounds described and/or depicted herein maybe provided and/or utilized in hydrate or solvate form.

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl. Furthermore, where a compound includes a plurality ofpositions at which substitutes are disclosed in groups or in ranges,unless otherwise indicated, the present disclosure is intended to coverindividual compounds and groups of compounds (e.g., genera andsubgenera) containing each and every individual subcombination ofmembers at each position.

Herein a phrase of the form “optionally substituted X” (e.g., optionallysubstituted alkyl) is intended to be equivalent to “X, wherein X isoptionally substituted” (e.g., “alkyl, wherein said alkyl is optionallysubstituted”). It is not intended to mean that the feature “X” (e.g.alkyl) per se is optional.

The term “alkyl,” as used herein, refers to saturated hydrocarbon groupscontaining from 1 to 20 (e.g., from 1 to 10 or from 1 to 6) carbons. Insome embodiments, an alkyl group is unbranched (i.e., is linear); insome embodiments, an alkyl group is branched. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxyl, optionally substituted with an O-protecting group; (9) nitro;(10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇ spirocyclyl; (12)thioalkoxy; (13) thiol; (14) —CO₂R^(A′), optionally substituted with anO-protecting group and where R^(A′) is selected from the groupconsisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₅₋₁₀aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₅₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₅₋₁₀ aryl, and (d) hydroxyl; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(E′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₅₋₁₀ aryl and (d) C₁₋₆alk-C₅₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₅₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₅₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (21)amidine; and (22) silyl groups such as trimethylsilyl,t-butyldimethylsilyl, and tri-isopropylsilyl. In some embodiments, eachof these groups can be further substituted as described herein. Forexample, the alkylene group of a C₁-alkaryl can be further substitutedwith an oxo group to afford the respective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionallysubstituted with an O-protecting group, such as optionally substitutedarylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl(e.g., acetyl, trifluoroacetyl, or others described herein),alkoxycarbonylalkyl (e.g., optionally substituted with an O-protectinggroup, such as optionally substituted arylalkoxycarbonyl groups or anydescribed herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl(e.g., alkheteroaryl), wherein each of these recited R^(N1) groups canbe optionally substituted, as defined herein for each group; or twoR^(N1) combine to form a heterocyclyl or an N-protecting group, andwherein each R^(N2) is, independently, H, alkyl, or aryl. The aminogroups of the invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2)2, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others describedherein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl) or aryl, andeach R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). As used herein, the term “amino acid” inits broadest sense, refers to any compound and/or substance that can beincorporated into a polypeptide chain, e.g., through formation of one ormore peptide bonds. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” refers to any of the twenty standard L-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.In some embodiments, an amino acid, including a carboxy- and/oramino-terminal amino acid in a polypeptide, can contain a structuralmodification as compared with the general structure above. For example,in some embodiments, an amino acid may be modified by methylation,amidation, acetylation, and/or substitution as compared with the generalstructure. In some embodiments, such modification may, for example,alter the circulating half life of a polypeptide containing the modifiedamino acid as compared with one containing an otherwise identicalunmodified amino acid. In some embodiments, such modification does notsignificantly alter a relevant activity of a polypeptide containing themodified amino acid, as compared with one containing an otherwiseidentical unmodified amino acid. As will be clear from context, in someembodiments, the term “amino acid” is used to refer to a free aminoacid; in some embodiments it is used to refer to an amino acid residueof a polypeptide. In some embodiments, the amino acid is attached to theparent molecular group by a carbonyl group, where the side chain oramino group is attached to the carbonyl group. In some embodiments, theamino acid is an α-amino acid. In certain embodiments, the amino acid isa β-amino acid. In some embodiments, the amino acid is a γ-amino acid.Exemplary side chains include an optionally substituted alkyl, aryl,heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, andcarboxyalkyl. Exemplary amino acids include alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine,norvaline, ornithine, phenylalanine, proline, pyrrolysine,selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, andvaline. Amino acid groups may be optionally substituted with one, two,three, or, in the case of amino acid groups of two carbons or more, foursubstituents independently selected from the group consisting of: (1)C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxyl;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of (CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each ofs2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H orC₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxyl; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(E′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₅₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₅₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₅₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxyl; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆thioalkoxy); (17) —(CH₂)_(g)CO₂R^(A′), where q is an integer from zeroto four, and R^(A′) is selected from the group consisting of (a) C₁₋₆alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(g)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(g)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(g)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(E′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The “arylalkyl” group, which as used herein, represents an aryl group,as defined herein, attached to the parent molecular group through analkylene group, as defined herein. Exemplary unsubstituted arylalkylgroups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20carbons, such as C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀alk-C₆₋₁₀ aryl). In some embodiments, the alkylene and the aryl each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective groups. Other groups preceded by the prefix“alk-” are defined in the same manner, where “alk” refers to a C₁₋₆alkylene, unless otherwise noted, and the attached chemical structure isas defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicnon-aromatic ring structure in which the rings are formed by carbonatoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, andcycloalkynyl groups.

The “carbocyclylalkyl” group, which as used herein, represents acarbocyclic group, as defined herein, attached to the parent moleculargroup through an alkylene group, as defined herein. Exemplaryunsubstituted carbocyclylalkyl groups are from 7 to 30 carbons (e.g.,from 7 to 16 or from 7 to 20 carbons, such as C₁₋₆ alk-C₆₋₁₀carbocyclyl, C₁₋₁₀ alk-C₆₋₁₀ carbocyclyl, or C₁₋₂₀ alk-C₆₋₁₀carbocyclyl). In some embodiments, the alkylene and the carbocyclyl eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective groups. Other groups preceded by theprefix “alk-” are defined in the same manner, where “alk” refers to aC₁₋₆ alkylene, unless otherwise noted, and the attached chemicalstructure is as defined herein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxy,” as used herein, means —CO₂H.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycle heptyl, andthe like. When the cycloalkyl group includes one carbon-carbon doublebond, the cycloalkyl group can be referred to as a “cycloalkenyl” group.Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, andthe like. The cycloalkyl groups of this invention can be optionallysubstituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₅₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxyl; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆thioalkoxy); (17) —(CH₂)_(g)CO₂R^(A′), where q is an integer from zeroto four, and R^(A′) is selected from the group consisting of (a) C₁₋₆alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₅₋₁₀ aryl; (18)—(CH₂)_(g)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₅₋₁₀ aryl; (19) —(CH₂)_(g)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(g)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₅₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The “cycloalkylalkyl” group, which as used herein, represents acycloalkyl group, as defined herein, attached to the parent moleculargroup through an alkylene group, as defined herein (e.g., an alkylenegroup of from 1 to 4, from 1 to 6, from 1 to 10, or form 1 to 20carbons). In some embodiments, the alkylene and the cycloalkyl each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “heteroalkyl,” as used herein, refers to an alkyl group, asdefined herein, in which one or two of the constituent carbon atoms haveeach been replaced by nitrogen, oxygen, or sulfur. In some embodiments,the heteroalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups. The terms“heteroalkenyl” and heteroalkynyl,” as used herein refer to alkenyl andalkynyl groups, as defined herein, respectively, in which one or two ofthe constituent carbon atoms have each been replaced by nitrogen,oxygen, or sulfur. In some embodiments, the heteroalkenyl andheteroalkynyl groups can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heteroarylalkyl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted heteroarylalkyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-1-12 heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxyl; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(g)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(g)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(g)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(g)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(E′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The “heterocyclylalkyl” group, which as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an alkylene group, as defined herein. Exemplaryunsubstituted heterocyclylalkyl groups are from 2 to 32 carbons (e.g.,from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15,from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as C₁₋₆alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂ heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂heterocyclyl). In some embodiments, the alkylene and the heterocyclyleach can be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxyl,” as used herein, represents an —OH group. In someembodiments, the hydroxyl group can be substituted with 1, 2, 3, or 4substituent groups (e.g., O-protecting groups) as defined herein for analkyl.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “O-protecting group,” as used herein, represents those groupsintended to protect an oxygen containing (e.g., phenol, hydroxyl, orcarbonyl) group against undesirable reactions during syntheticprocedures. Commonly used O-protecting groups are disclosed in Greene,“Protective Groups in Organic Synthesis,” 3^(rd) Edition (John Wiley &Sons, New York, 1999), which is incorporated herein by reference.Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups,such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl,tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl,phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and4-nitrobenzoyl; alkylcarbonyl groups, such as acyl, acetyl, propionyl,pivaloyl, and the like; optionally substituted arylcarbonyl groups, suchas benzoyl; silyl groups, such as trimethylsilyl (TMS),tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM),triisopropylsilyl (TIPS), and the like; ether-forming groups with thehydroxyl, such methyl, methoxymethyl, tetrahydropyranyl, benzyl,p-methoxybenzyl, trityl, and the like; alkoxycarbonyls, such asmethoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl,sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl,cyclohexyloxycarbonyl, methyloxycarbonyl, and the like;alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl,2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl,allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl,3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls, such as2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, and the like; optionally substitutedarylalkoxycarbonyl groups, such as benzyloxycarbonyl,p-methylbenzyloxycarbonyl, p methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl,3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxy-carbonyl, fluorenylmethyloxycarbonyl, and the like; andoptionally substituted aryloxycarbonyl groups, such as phenoxycarbonyl,p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl,2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl,m-methylphenoxycarbonyl, o-bromophenoxycarbonyl,3,5-dimethylphenoxycarbonyl, p chlorophenoxycarbonyl,2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl,aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl; methoxymethyl;benzyloxymethyl; siloxymethyl; 2,2,2-trichloroethoxymethyl;tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl;1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether;p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl,and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; anddiphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl;vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl;and nitrobenzyl); carbonyl-protecting groups (e.g., acetal and ketalgroups, such as dimethyl acetal, 1,3-dioxolane, and the like; acylalgroups; and dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, andthe like); carboxylic acid-protecting groups (e.g., ester groups, suchas methyl ester, benzyl ester, t-butyl ester, orthoesters, and the like;and oxazoline groups.

The term “oxo” as used herein, represents ═O.

The prefix “perfluoro,” as used herein, represents anyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. For example, perfluoroalkyl groupsare exemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “protected hydroxyl,” as used herein, refers to an oxygen atombound to an O-protecting group.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thiol,” as used herein, represents an —SH group.

Definitions

In this application, unless otherwise clear from context, (i) the term“a” may be understood to mean “at least one”; (ii) the term “or” may beunderstood to mean “and/or”; (iii) the terms “comprising” and“including” may be understood to encompass itemized components or stepswhether presented by themselves or together with one or more additionalcomponents or steps; and (iv) the terms “about” and “approximately” maybe understood to permit standard variation as would be understood bythose of ordinary skill in the art; and (v) where ranges are provided,endpoints are included.

As used herein, the term “π-effect interaction” refers to attractive,non-covalent interactions between aromatic rings.

As used herein, the term “active site” refers to the location on aprotein (e.g., an enzyme) where substrate molecules bind and undergo achemical reaction. By “does not bind at the active site” is meant thatno atoms of a compound or complex substantially participate in bindingwith residues within the active site (e.g., residues that participate inbinding to a natural substrate molecule).

As used herein, the term “administration” refers to the administrationof a composition (e.g., a compound, a complex or a preparation thatincludes a compound or complex as described herein) to a subject orsystem. Administration to an animal subject (e.g., to a human) may be byany appropriate route. For example, in some embodiments, administrationmay be bronchial (including by bronchial instillation), buccal, enteral,interdermal, intra-arterial, intradermal, intragastric, intramedullary,intramuscular, intranasal, intraperitoneal, intrathecal, intravenous,intraventricular, mucosal, nasal, oral, rectal, subcutaneous,sublingual, topical, tracheal (including by intratracheal instillation),transdermal, vaginal and vitreal.

As is known in the art, “affinity” is a measure of the tightness withwhich a particular ligand binds to its partner. Affinities can bemeasured in different ways. In some embodiments, affinity is measured bya quantitative assay. In some such embodiments, binding partnerconcentration may be fixed to be in excess of ligand concentration so asto mimic physiological conditions. Alternatively or additionally, insome embodiments, binding partner concentration and/or ligandconcentration may be varied. In some such embodiments, affinity may becompared to a reference under comparable conditions (e.g.,concentrations).

As used herein, the term “analog” refers to a substance that shares oneor more particular structural features, elements, components, ormoieties with a reference substance. Typically, an “analog” showssignificant structural similarity with the reference substance, forexample sharing a core or consensus structure, but also differs incertain discrete ways. In some embodiments, an analog is a substancethat can be generated from the reference substance by chemicalmanipulation of the reference substance. In some embodiments, an analogis a substance that can be generated through performance of a syntheticprocess substantially similar to (e.g., sharing a plurality of stepswith) one that generates the reference substance. In some embodiments,an analog is or can be generated through performance of a syntheticprocess different from that used to generate the reference substance.

As used herein, the term “animal” refers to any member of the animalkingdom. In some embodiments, “animal” refers to humans, at any stage ofdevelopment. In some embodiments, “animal” refers to non-human animals,at any stage of development. In some embodiments, the non-human animalis a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog,a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments,animals include, but are not limited to, mammals, birds, reptiles,amphibians, fish, and/or worms. In some embodiments, an animal may be atransgenic animal, genetically-engineered animal, and/or a clone.

As used herein, the term “antagonist” refers to a compound that i)inhibits, decreases or reduces the effects of CEP250; and/or ii)inhibits, decreases, reduces, or delays one or more biological events.An antagonist may be direct (in which case it exerts its influencedirectly upon its target) or indirect (in which case it exerts itsinfluence by other than binding to its target; e.g., by interacting witha regulator of CEP250, for example so that level or activity of CEP250is altered).

As used herein, the terms “approximately” and “about” are each intendedto encompass normal statistical variation as would be understood bythose of ordinary skill in the art as appropriate to the relevantcontext. In certain embodiments, the terms “approximately” or “about”each refer to a range of values that fall within 25%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or less in either direction (greater than or less than) of a statedvalue, unless otherwise stated or otherwise evident from the context(e.g., where such number would exceed 100% of a possible value).

Two events or entities are “associated” with one another, as that termis used herein, if the presence, level and/or form of one is correlatedwith that of the other. For example, a particular entity (e.g.,polypeptide) is considered to be associated with a particular disease,disorder, or condition, if its presence, level and/or form correlateswith incidence of and/or susceptibility of the disease, disorder, orcondition (e.g., across a relevant population). In some embodiments, twoor more entities are physically “associated” with one another if theyinteract, directly or indirectly, so that they are and remain inphysical proximity with one another. In some embodiments, two or moreentities that are physically associated with one another are covalentlylinked to one another; in some embodiments, two or more entities thatare physically associated with one another are not covalently linked toone another but are non-covalently associated, for example by means ofhydrogen bonds, van der Waals interaction, hydrophobic interactions,magnetism, and combinations thereof.

It will be understood that the term “binding” as used herein, typicallyrefers to association (e.g., non-covalent or covalent) between or amongtwo or more entities. “Direct” binding involves physical contact betweenentities or moieties; indirect binding involves physical interaction byway of physical contact with one or more intermediate entities. Bindingbetween two or more entities can typically be assessed in any of avariety of contexts—including where interacting entities or moieties arestudied in isolation or in the context of more complex systems (e.g.,while covalently or otherwise associated with a carrier entity and/or ina biological system or cell).

The affinity of a molecule X for its partner Y can generally berepresented by the dissociation constant (K_(D)). Affinity can bemeasured by common methods known in the art, including those describedherein. Specific illustrative and exemplary embodiments for measuringbinding affinity are described below. The term “K_(D),” as used herein,is intended to refer to the dissociation equilibrium constant of aparticular compound-protein or complex-protein interaction. Typically,the compounds of the invention bind to presenter proteins with adissociation equilibrium constant (K_(D)) of less than about 10⁻⁶ M,such as less than approximately 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M oreven lower, e.g., when determined by surface plasmon resonance (SPR)technology using the presenter protein as the analyte and the compoundas the ligand. The presenter protein/compound complexes of the inventionbind to CEP250 with a dissociation equilibrium constant (K_(D)) of lessthan about 10⁻⁶ M, such as less than approximately 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹M, or 10⁻¹⁰ M or even lower, e.g., when determined by surface plasmonresonance (SPR) technology using CEP250 as the analyte and the complexas the ligand.

As used herein, the term “binding free energy” refers to the differencein energy between bound and unbound states of a complex. Binding freeenergy may be determined by methods known in the art including using theequation ΔG=−RTInK using an experimentally derived dissociationconstant, Kd. Binding free energy can be calculated using acomputational algorithm known in the art, e.g., molecular dynamicsimulations, free-energy perturbations or Monte Carlo protocols, asimplemented in commercial software such as AMBER (Cornell et al. J. Am.Chem. Soc. 1995, 117, 5179) CHARMM (Brooks et al. J. Comp. Chem. 1983,4, 187), or Desmond (Boowers et al. Proc. ACM/IEEE Conf. Supercomputing,2006, SCO6).

As used herein, the term “buried surface area” refers to the surfacearea of a protein or a complex that is not exposed to solvent. Buriedsurface area may be determined by methods known in the art including bycalculating inaccessibility to solvent. Inaccessibility to solvent maybe calculated computationally with a rolling probe of 1.4 Å, using aprogram such as PDBePISA release version 1.48(http://www.ebi.ac.uk/pdbe/pisa/).

As used herein, the term “cell penetrant” refers to compounds that whenadded to a cell's environment enter the intracellular domain withoutkilling the cell. Whether a compound is cell penetrant may be determinedusing any method known in the art, e.g., the biosensor method describedherein

As used herein, the term “characteristic portion” is used, in thebroadest sense, to refer to a portion of a substance whose presence (orabsence) correlates with presence (or absence) of a particular feature,attribute, or activity of the substance. In some embodiments, acharacteristic portion of a substance is a portion that is found in thesubstance and in related substances that share the particular feature,attribute or activity, but not in those that do not share the particularfeature, attribute or activity. In certain embodiments, a characteristicportion shares at least one functional characteristic with the intactsubstance. For example, in some embodiments, a “characteristic portion”of a protein or polypeptide is one that contains a continuous stretch ofamino acids, or a collection of continuous stretches of amino acids,that together are characteristic of a protein or polypeptide. In someembodiments, each such continuous stretch generally contains at least 2,5, 10, 15, 20, 50, or more amino acids. In general, a characteristicportion of a substance (e.g., of a protein, antibody, etc.) is one that,in addition to the sequence and/or structural identity specified above,shares at least one functional characteristic with the relevant intactsubstance. In some embodiments, a characteristic portion may bebiologically active.

As used herein, the phrase “characteristic sequence element” refers to asequence element found in a polymer (e.g., in a polypeptide or nucleicacid) that represents a characteristic portion of that polymer. In someembodiments, presence of a characteristic sequence element correlateswith presence or level of a particular activity or property of thepolymer. In some embodiments, presence (or absence) of a characteristicsequence element defines a particular polymer as a member (or not amember) of a particular family or group of such polymers. Acharacteristic sequence element typically comprises at least twomonomers (e.g., amino acids or nucleotides). In some embodiments, acharacteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers(e.g., contiguously linked monomers). In some embodiments, acharacteristic sequence element includes at least first and secondstretches of contiguous monomers spaced apart by one or more spacerregions whose length may or may not vary across polymers that share thesequence element. In certain embodiments, particular characteristicsequence elements may be referred to as “motifs”.

As used herein, the term “clogP” refers to the calculated partitioncoefficient of a molecule or portion of a molecule. The partitioncoefficient is the ratio of concentrations of a compound in a mixture oftwo immiscible phases at equilibrium (e.g., octanol and water) andmeasures the hydrophobicity or hydrophilicity of a compound. A varietyof methods are available in the art for determining clogP. For example,in some embodiments, clogP can be determined using quantitativestructure-property relationship algorithims known in the art (e.g.,using fragment based prediction methods that predict the log P of acompound by determining the sum of its non-overlapping molecularfragments). Several algorithims for calculating clog P are known in theart including those used by molecular editing software such as CHEMDRAW®Pro, Version 12.0.2.1092 (Camrbridgesoft, Cambridge, Mass.) andMARVINSKETCH® (ChemAxon, Budapest, Hungary). A compound is considered tohave met a threshold c Log P if it meets the threshold in at least oneof the above methods.

As used herein, the term “collection” refers to a group of 2, 5, 10,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or more different molecules. Insome embodiments, at least 10% (e.g., at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99% or 100%) of the compounds in thecollection are compounds (e.g., macrocyclic compounds)s describedherein.

As used herein, the term “combination therapy” refers to thosesituations in which a subject is simultaneously exposed to two or moretherapeutic regimens (e.g., two or more compounds such as macrocycliccompounds). In some embodiments, two or more compounds may beadministered simultaneously; in some embodiments, such compounds may beadministered sequentially; in some embodiments, such compounds areadministered in overlapping dosing regimens.

The term “comparable,” as used herein, refers to two or more compounds,entities, situations, sets of conditions, etc that may not be identicalto one another but that are sufficiently similar to permit comparisontherebetween so that conclusions may reasonably be drawn based ondifferences or similarities observed. In some embodiments, comparablesets of conditions, circumstances, individuals, or populations arecharacterized by a plurality of substantially identical features and oneor a small number of varied features. Those of ordinary skill in the artwill understand, in context, what degree of identity is required in anygiven circumstance for two or more such compounds, entities, situations,sets of conditions, etc to be considered comparable. For example, thoseof ordinary skill in the art will appreciate that sets of circumstances,individuals, or populations are comparable to one another whencharacterized by a sufficient number and type of substantially identicalfeatures to warrant a reasonable conclusion that differences in resultsobtained or phenomena observed under or with different sets ofcircumstances, individuals, or populations are caused by or indicativeof the variation in those features that are varied.

As used herein, the term “complex” refers to a group of two or morecompounds and/or proteins which are bound together through a bindinginteraction (e.g., a non-covalent interaction, such as a hydrophobiceffect interaction, an electrostatic interaction, a van der Waalsinteraction, or π-effect interaction). Examples of complexes are“presenter protein/compound complex” which include a compound of theinvention bound to a presenter protein.

As used herein, the term “corresponding to” is often used to designate astructural element or moiety in an compound of interest that shares aposition (e.g., in three-dimensional space or relative to anotherelement or moiety) with one present in an appropriate referencecompound. For example, in some embodiments, the term is used to refer toposition/identity of a residue in a polymer, such as an amino acidresidue in a polypeptide or a nucleotide residue in a nucleic acid.Those of ordinary skill will appreciate that, for purposes ofsimplicity, residues in such a polymer are often designated using acanonical numbering system based on a reference related polymer, so thata residue in a first polymer “corresponding to” a residue at position190 in the reference polymer, for example, need not actually be the190th residue in the first polymer but rather corresponds to the residuefound at the 190th position in the reference polymer; those of ordinaryskill in the art readily appreciate how to identify “corresponding”amino acids, including through use of one or more commercially-availablealgorithms specifically designed for polymer sequence comparisons.

As used herein, the term “designed” refers to a compound (i) whosestructure is or was selected by the hand of man; (ii) that is producedby a process requiring the hand of man; and/or (iii) that is distinctfrom natural substances and other known compounds.

Many methodologies described herein include a step of “determining.”Those of ordinary skill in the art, reading the present specification,will appreciate that such “determining” can utilize or be accomplishedthrough use of any of a variety of techniques available to those skilledin the art, including for example specific techniques explicitlyreferred to herein. In some embodiments, determining involvesmanipulation of a physical sample. In some embodiments, determininginvolves consideration and/or manipulation of data or information, forexample utilizing a computer or other processing unit adapted to performa relevant analysis. In some embodiments, determining involves receivingrelevant information and/or materials from a source. In someembodiments, determining involves comparing one or more features of asample or entity to a comparable reference.

As used herein, the term “depsi-linkage” refers to the replacement of anamide bond with an ester bond.

As used herein, the term “dosage form” refers to a physically discreteunit of an active compound (e.g., a therapeutic or diagnostic agent) foradministration to a subject. Each unit contains a predetermined quantityof active agent. In some embodiments, such quantity is a unit dosageamount (or a whole fraction thereof) appropriate for administration inaccordance with a dosing regimen that has been determined to correlatewith a desired or beneficial outcome when administered to a relevantpopulation (i.e., with a therapeutic dosing regimen). Those of ordinaryskill in the art appreciate that the total amount of a therapeuticcomposition or compound administered to a particular subject isdetermined by one or more attending physicians and may involveadministration of multiple dosage forms.

As used herein, the term “dosing regimen” refers to a set of unit doses(typically more than one) that are administered individually to asubject, typically separated by periods of time. In some embodiments, agiven therapeutic compound has a recommended dosing regimen, which mayinvolve one or more doses. In some embodiments, a dosing regimencomprises a plurality of doses each of which are separated from oneanother by a time period of the same length; in some embodiments, adosing regimen comprises a plurality of doses and at least two differenttime periods separating individual doses. In some embodiments, all doseswithin a dosing regimen are of the same unit dose amount. In someembodiments, different doses within a dosing regimen are of differentamounts. In some embodiments, a dosing regimen comprises a first dose ina first dose amount, followed by one or more additional doses in asecond dose amount different from the first dose amount. In someembodiments, a dosing regimen comprises a first dose in a first doseamount, followed by one or more additional doses in a second dose amountsame as the first dose amount In some embodiments, a dosing regimen iscorrelated with a desired or beneficial outcome when administered acrossa relevant population (i.e., is a therapeutic dosing regimen).

As used herein, the term “engineered” is used to describe a compoundwhose design and/or production involves action of the hand of man. Forexample, in some embodiments, an “engineered” compound is prepared by invitro chemical synthesis. In some embodiments, an “engineered” compoundis produced by a cell that has been genetically manipulated relative toa reference wild type cell. In some embodiments, an “engineered”compound is produced by a cell in culture. In some embodiments, an“engineered” compound is produced by a cell in culture conditionsspecifically modified to enhance production of the compound. In someembodiments, an “engineered” compound has a structure designed orselected by in silico modeling.

As used herein, the term “flat surface site,” as is understood in theart, refers to a site on a surface of a protein structure that has arelatively flat character, (e.g., a site that does not include awell-defined pocket or cavity with an area of greater than 500 Å², avolume of greater than 400 Å³, and a depth greater than 13 Å). In someembodiments, a site may be determined to be flat by utilizing acommercial algorithm known in the art e.g., a site may be determined tobe flat if it does not include a well-defined pocket or cavity with anarea of greater than 500 Å², a volume of greater than 400 Å₃, and adepth greater than 13 Å as determined by CAST (Liang et al. Prot. Sci.1998, 7, 1884) or Sitemap (Halgren J. Chem. Inf. Model. 2009, 49, 377).Those of oridinary skill in the art are familiar with the concept offlatness and, moreover are aware of its relationship to “druggability.”In some embodiments, a protein is considered to have a flat surface siteif it is undruggable as defined herein, e.g., is determined to beundruggable using the program DOGSITESCORER®.

As used herein, the term “hydrophobic residue” refers to an amino acidthat has a hydropathy index value equal to or greater than proline.Examples of hydrophobic residues are valine, isoleucine, leucine,methionine, phenylalanine, tryptophan, alanine, glycine, and cysteine.

As used herein, the term “hydrophobic surface site” refers to site on asurface of a protein structure comprising at least 30% hydrophobicresidues).

As used herein, the term “identity” refers to the overall relatednessbetween polymeric molecules, e.g., between nucleic acid molecules (e.g.,DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. Calculation of the percent identity of two nucleic acidsequences, for example, can be performed by aligning the two sequencesfor optimal comparison purposes (e.g., gaps can be introduced in one orboth of a first and a second nucleic acid sequences for optimalalignment and non identical sequences can be disregarded for comparisonpurposes). In certain embodiments, the length of a sequence aligned forcomparison purposes is at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, orsubstantially 100% of the length of the reference sequence. Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleotide sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.

As used herein, the term “isolated” refers to a substance and/or entitythat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in natureand/or in an experimental setting), and/or (2) designed, produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedcompounds are about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or more than about 99% pure. As used herein, a substance is “pure”if it is substantially free of other components. In some embodiments, aswill be understood by those skilled in the art, a substance may still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. In some embodiments, isolationinvolves or requires disruption of covalent bonds (e.g., to isolate apolypeptide domain from a longer polypeptide and/or to isolate anucleotide sequence element from a longer oligonucleotide or nucleicacid).

The term “macrocyclic compound,” as used herein, refers to a smallmolecule compound containing a ring with nine or more ring atoms.Macrocyclic compounds include marcrolides, a group of small moleculescontaining a macrocyclic lactone, such as erythromycin, rapamycin, andFK506. In some embodiments, a macrocyclic compound is a small moleculein which greater than 25% (e.g., greater than 30%, greater than 35%,greater than 40%, greater than 45%) of the non-hydrogen atoms in thesmall molecule are included in a single or fused ring structure. In someembodiments, the macrocyclic compound is not a compound described inBenjamin et al. Nat. Rev. Drug. Discov. 2011, 10(11), 868-880 orSweeney, Z. K. et al. J. Med. Chem. 2014, epub ahead of print, thestructures of which are incorporated by reference.

The term “modulator” is used to refer to an entity whose presence orlevel in a system in which an activity of interest is observedcorrelates with a change in level and/or nature of that activity ascompared with that observed under otherwise comparable conditions whenthe modulator is absent. In some embodiments, a modulator is anactivator, in that activity is increased in its presence as comparedwith that observed under otherwise comparable conditions when themodulator is absent. In some embodiments, a modulator is an antagonistor inhibitor, in that activity is reduced in its presence as comparedwith otherwise comparable conditions when the modulator is absent. Insome embodiments, a modulator interacts directly with a target entitywhose activity is of interest. In some embodiments, a modulatorinteracts indirectly (i.e., directly with an intermediate compound thatinteracts with the target entity) with a target entity whose activity isof interest. In some embodiments, a modulator affects level of a targetentity of interest; alternatively or additionally, in some embodiments,a modulator affects activity of a target entity of interest withoutaffecting level of the target entity. In some embodiments, a modulatoraffects both level and activity of a target entity of interest, so thatan observed difference in activity is not entirely explained by orcommensurate with an observed difference in level. In some embodiments,a modulator is an allosteric modulator such as an allosteric agonist.

The term “N-alkylated amino acids” as used herein, refers to amino acidscontaining an optionally substituted C₁ to C₆ alkyl on the nitrogen ofthe amino acid that forms the peptidic bond. N-alkylated amino acidsinclude, but are not limited to, N-methyl amino acids, such asN-methyl-alanine, N-methyl-threonine, N-methyl-phenylalanine,N-methyl-aspartic acid, N-methyl-valine, N-methyl-leucine,N-methyl-glycine, N-methyl-isoleucine, N(α)-methyl-lysine,N(α)-methyl-asparagine, and N(α)-methyl-glutamine.

As used herein, an atom that “participates in binding” is within 4 Å ofthe entity to which they bind or connects to an atom that is with 4 Å ofthe entity to which they bind.

As used herein, the term “pharmaceutical composition” refers to anactive compound, formulated together with one or more pharmaceuticallyacceptable carriers. In some embodiments, active compound is present inunit dose amount appropriate for administration in a therapeutic regimenthat shows a statistically significant probability of achieving apredetermined therapeutic effect when administered to a relevantpopulation. In some embodiments, pharmaceutical compositions may bespecially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

A “pharmaceutically acceptable excipient,” as used herein, refers anyinactive ingredient (for example, a vehicle capable of suspending ordissolving the active compound) having the properties of being nontoxicand non-inflammatory in a subject. Typical excipients include, forexample: antiadherents, antioxidants, binders, coatings, compressionaids, disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspensing or dispersing agents, sweeteners, or waters of hydration.Excipients include, but are not limited to: butylated hydroxytoluene(BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate,croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid,crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate,maltitol, mannitol, methionine, methylcellulose, methyl paraben,microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone,povidone, pregelatinized starch, propyl paraben, retinyl palmitate,shellac, silicon dioxide, sodium carboxymethyl cellulose, sodiumcitrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid,stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E,vitamin C, and xylitol. Those of ordinary skill in the art are familiarwith a variety of agents and materials useful as excipients.

The term “pharmaceutically acceptable salt,” as use herein, refers tothose salts of the compounds described here that are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof humans and animals without undue toxicity, irritation, allergicresponse and the like and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art. For example, pharmaceutically acceptable salts are describedin: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and inPharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahland C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situduring the final isolation and purification of the compounds describedherein or separately by reacting the free base group with a suitableorganic acid.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be acid addition salts involving inorganic or organic acids or thesalts may, in the case of acidic forms of the compounds of the inventionbe prepared from inorganic or organic bases. Frequently, the compoundsare prepared or used as pharmaceutically acceptable salts prepared asaddition products of pharmaceutically acceptable acids or bases.Suitable pharmaceutically acceptable acids and bases are well-known inthe art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic,citric, or tartaric acids for forming acid addition salts, and potassiumhydroxide, sodium hydroxide, ammonium hydroxide, caffeine, variousamines, and the like for forming basic salts. Methods for preparation ofthe appropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2 hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts andthe like. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamineand the like.

The term “polar surface area” refers to the surface sum over all polaratoms of a molecule or portion of a molecule, including their attachedhydrogens. Polar surface area is determined computationally using aprogram such as CHEMDRAW® Pro, Version 12.0.2.1092 (Cambridgesoft,Cambridge, Mass.).

The term “presenter protein” refers to a protein that binds to a smallmolecule to form a complex that binds to and modulates the activity ofCEP250. In some embodiments, the presenter protein is a relativelyabundant protein (e.g., the presenter protein is sufficiently abundantthat participation in a tripartite complex does not substantially impactthe biological role of the presenter protein in a cell and/or viabilityor other attributes of the cell). In certain embodiments, the presenterprotein is a protein that has chaperone activity within a cell. In someembodiments, the presenter protein is a protein that has multiplenatural interaction partners within a cell. In certain embodiments, thepresenter protein is one which is known to bind a small molecule to forma binary complex that is known to or suspected of binding to andmodulating the biological activity of CEP250.

The term “presenter protein binding moiety” refers to a group of ringatoms and the moieties attached thereto (e.g., atoms within 20 atoms ofa ring atom such as, atoms within 15 atoms of a ring atom, atoms within10 atoms of a ring atom, atoms within 5 atoms of a ring atom) thatparticipate in binding to a presenter protein such that the compoundspecifically binds to said presenter protein, for example, with a K_(D)of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50nM, less than 25 nM, less than 10 nM) or inhibits the peptidyl-prolylisomerase activity of the presenter protein, for example, with an IC₅₀of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than0.05 μM, less than 0.01 μM). It will be understood that the presenterprotein binding moiety does not necessarily encompass the entirety ofatoms in the compound that interact with the presenter protein. It willalso be understood that one or more atoms of the presenter proteinbinding moiety may be within the CEP250 interaction moiety.

The term “pure” means substantially pure or free of unwanted components(e.g., other compounds and/or other components of a cell lysate),material defilement, admixture or imperfection.

The term “reference” is often used herein to describe a standard orcontrol compound, individual, population, sample, sequence or valueagainst which a compound, individual, population, sample, sequence orvalue of interest is compared. In some embodiments, a referencecompound, individual, population, sample, sequence or value is testedand/or determined substantially simultaneously with the testing ordetermination of the compound, individual, population, sample, sequenceor value of interest. In some embodiments, a reference compound,individual, population, sample, sequence or value is a historicalreference, optionally embodied in a tangible medium. Typically, as wouldbe understood by those skilled in the art, a reference compound,individual, population, sample, sequence or value is determined orcharacterized under conditions comparable to those utilized to determineor characterize the compound, individual, population, sample, sequenceor value of interest.

The term “ring atoms” refers to the atoms of a cyclic compound thatcomprise the innermost portion of the ring. For example, using thismethod FK506 has 21 ring atoms and rapamycin has 29 ring atoms.

The term “site of a naturally occurring protein-protein interaction”refers to a location on the surface of the structure of a protein thatincludes atoms which participate in binding between the protein andanother protein in the proteins natural environment.

The term “small molecule” means a low molecular weight organic and/orinorganic compound. In general, a “small molecule” is a molecule that isless than about 5 kilodaltons (kD) in size. In some embodiments, a smallmolecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. Insome embodiments, the small molecule is less than about 800 daltons (D),about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, orabout 100 D. In some embodiments, a small molecule is less than about2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, lessthan about 800 g/mol, or less than about 500 g/mol. In some embodiments,a small molecule is not a polymer. In some embodiments, a small moleculedoes not include a polymeric moiety. In some embodiments, a smallmolecule is not a protein or polypeptide (e.g., is not an oligopeptideor peptide). In some embodiments, a small molecule is not apolynucleotide (e.g., is not an oligonucleotide). In some embodiments, asmall molecule is not a polysaccharide. In some embodiments, a smallmolecule does not comprise a polysaccharide (e.g., is not aglycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, asmall molecule is not a lipid. In some embodiments, a small molecule isa modulating compound. In some embodiments, a small molecule isbiologically active. In some embodiments, a small molecule is detectable(e.g., comprises at least one detectable moiety). In some embodiments, asmall molecule is a therapeutic.

Those of ordinary skill in the art, reading the present disclosure, willappreciate that certain small molecule compounds described herein may beprovided and/or utilized in any of a variety of forms such as, forexample, salt forms, protected forms, pro-drug forms, ester forms,isomeric forms (e.g., optical and/or structural isomers), isotopicforms, etc. In some embodiments, reference to a particular compound mayrelate to a specific form of that compound. In some embodiments,reference to a particular compound may relate to that compound in anyform. In some embodiments, where a compound is one that exists or isfound in nature, that compound may be provided and/or utilized inaccordance in the present invention in a form different from that inwhich it exists or is found in nature. Those of ordinary skill in theart will appreciate that a compound preparation including a differentlevel, amount, or ratio of one or more individual forms than a referencepreparation or source (e.g., a natural source) of the compound may beconsidered to be a different form of the compound as described herein.Thus, in some embodiments, for example, a preparation of a singlestereoisomer of a compound may be considered to be a different form ofthe compound than a racemic mixture of the compound; a particular saltof a compound may be considered to be a different form from another saltform of the compound; a preparation containing one conformational isomer((Z) or (E)) of a double bond may be considered to be a different formfrom one containing the other conformational isomer ((E) or (Z)) of thedouble bond; a preparation in which one or more atoms is a differentisotope than is present in a reference preparation may be considered tobe a different form; etc.

As used herein, the terms “specific binding” or “specific for” or“specific to” refer to an interaction between a binding agent and atarget entity. As will be understood by those of ordinary skill, aninteraction is considered to be “specific” if it is favored in thepresence of alternative interactions, for example, binding with a K_(D)of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50nM, less than 25 nM, less than 10 nM). In many embodiments, specificinteraction is dependent upon the presence of a particular structuralfeature of the target entity (e.g., an epitope, a cleft, a bindingsite). It is to be understood that specificity need not be absolute. Insome embodiments, specificity may be evaluated relative to that of thebinding agent for one or more other potential target entities (e.g.,competitors). In some embodiments, specificity is evaluated relative tothat of a reference specific binding agent. In some embodimentsspecificity is evaluated relative to that of a reference non-specificbinding agent.

The term “specific” when used with reference to a compound having anactivity, is understood by those skilled in the art to mean that thecompound discriminates between potential target entities or states. Forexample, an in some embodiments, a compound is said to bind“specifically” to its target if it binds preferentially with that targetin the presence of one or more competing alternative targets. In manyembodiments, specific interaction is dependent upon the presence of aparticular structural feature of the target entity (e.g., an epitope, acleft, a binding site). It is to be understood that specificity need notbe absolute. In some embodiments, specificity may be evaluated relativeto that of the binding agent for one or more other potential targetentities (e.g., competitors). In some embodiments, specificity isevaluated relative to that of a reference specific binding agent. Insome embodiments specificity is evaluated relative to that of areference non-specific binding agent. In some embodiments, the agent orentity does not detectably bind to the competing alternative targetunder conditions of binding to its target entity. In some embodiments,binding agent binds with higher on-rate, lower off-rate, increasedaffinity, decreased dissociation, and/or increased stability to itstarget entity as compared with the competing alternative target(s).

The term “structural organization” refers to the average threedimensional configuration of the atoms and bonds of molecule. By“substantially unchanged structural organization” is meant that the rootmean squared deviation (RMSD) of two aligned structures is less than 1.The RMSD can be calculated, e.g., by using the align command in PyMOLversion 1.7rc1 (Schrödinger LLC). Alternatively, RMSD can be calculatedusing the ExecutiveRMS parameter from the algorithm LigAlign (J. Mol.Graphics and Modelling 2010, 29, 93-101).

The term “substantially” refers to the qualitative condition ofexhibiting total or near-total extent or degree of a characteristic orproperty of interest. One of ordinary skill in the biological arts willunderstand that biological and chemical phenomena rarely, if ever, go tocompletion and/or proceed to completeness or achieve or avoid anabsolute result. The term “substantially” is therefore used herein tocapture the potential lack of completeness inherent in many biologicaland chemical phenomena.

The term “does not substantially bind” to a particular protein as usedherein can be exhibited, for example, by a molecule or portion of amolecule having a K_(D) for the target of 10⁻⁴ M or greater,alternatively 10⁻⁵ M or greater, alternatively 10⁻⁶ M or greater,alternatively 10⁻⁷ M or greater, alternatively 10⁻⁸ M or greater,alternatively 10⁻⁹ M or greater, alternatively 10⁻¹⁰ M or greater,alternatively 10⁻¹¹ M or greater, alternatively 10⁻¹² M or greater, or aK_(D) in the range of 10⁻⁴ M to 10⁻¹² M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ Mto 10⁻⁹ M.

The term “substantial structural similarity” refers to presence ofshared structural features such as presence and/or identity ofparticular amino acids at particular positions (see definitions of“shared sequence homology” and “shared sequence identity”). In someembodiments the term “substantial structural similarity” refers topresence and/or identity of structural elements (for example: loops,sheets, helices, H-bond donors, H-bond acceptors, glycosylationpatterns, salt bridges, and disulfide bonds). In some some embodiments,the term “substantial structural similarity” refers to three dimensionalarrangement and/or orientation of atoms or moieties relative to oneanother (for example: distance and/or angles between or among thembetween an agent of interest and a reference agent).

The term “target protein” refers to a protein that is not mTOR orcalcineurin that binds with a small molecule/presenter protein/compoundcomplex as described herein, but that does not substantially bind witheither the small molecule or the presenter protein alone. In someembodiments, the small molecule/presenter protein/compound complex doesnot substantially bind to mTOR or calcineurin. In some embodiments, thetarget protein participates in a biological pathway associated with adisease, disorder or condition. In some embodiments, a target protein isa naturally-occurring protein; in some such embodiments, a targetprotein is naturally found in certain mammalian cells (e.g., a mammaliantarget protein), fungal cells (e.g., a fungal target protein), bacterialcells (e.g., a bacterial target protein) or plant cells (e.g., a planttarget protein). In some embodiments, a target protein is characterizedby natural interaction with one or more natural presenterprotein/natural small molecule complexes. In some embodiments, a targetprotein is characterized by natural interactions with a plurality ofdifferent natural presenter protein/natural small molecule complexes; insome such embodiments some or all of the complexes utilize the samepresenter protein (and different small molecules). In some embodiments,a target protein does not substantially bind to a complex ofcyclosporin, rapamycin, or FK506 and a presenter protein (e.g., FKBP).Target proteins can be naturally occurring, e.g., wild type.Alternatively, the target protein can vary from the wild type proteinbut still retain biological function, e.g., as an allelic variant, asplice mutant or a biologically active fragment.

The term “target protein interacting moiety” refers to a group of ringatoms and the moieties attached thereto (e.g., atoms within 20 atoms ofa ring atom such as, atoms within 15 atoms of a ring atom, atoms within10 atoms of a ring atom, atoms within 5 atoms of a ring atom) thatparticipate in binding to a target protein (e.g., a eukaryotic targetprotein such as a mammalian target protein or a fungal target protein ora prokaryotic target protein such as a bacterial target protein) whenthe compound is in a complex with a presenter protein. It will beunderstood that the target protein interacting moiety does notnecessarily encompass the entirety of atoms in the compound thatinteract with the target protein. It will also be understood that one ormore atoms of the presenter protein binding moiety may also be presentin the target protein interacting moiety.

The term “CEP250 interacting moiety” refers to a group of ring atoms andthe moieties attached thereto (e.g., atoms within 20 atoms of a ringatom such as, atoms within 15 atoms of a ring atom, atoms within 10atoms of a ring atom, atoms within 5 atoms of a ring atom) thatparticipate in binding to CEP250 when the compound is in a complex witha presenter protein. It will be understood that the CEP250 interactingmoiety does not necessarily encompass the entirety of atoms in thecompound that interact with CEP250. It will also be understood that oneor more atoms of the presenter protein binding moiety may also bepresent in the CEP250 protein interacting moiety.

A “therapeutic regimen” refers to a dosing regimen whose administrationacross a relevant population is correlated with a desired or beneficialtherapeutic outcome.

The term “therapeutically effective amount” means an amount that issufficient, when administered to a population suffering from orsusceptible to a disease, disorder, and/or condition in accordance witha therapeutic dosing regimen, to treat the disease, disorder, and/orcondition. In some embodiments, a therapeutically effective amount isone that reduces the incidence and/or severity of, and/or delays onsetof, one or more symptoms of the disease, disorder, and/or condition.Those of ordinary skill in the art will appreciate that the term“therapeutically effective amount” does not in fact require successfultreatment be achieved in a particular individual. Rather, atherapeutically effective amount may be that amount that provides aparticular desired pharmacological response in a significant number ofsubjects when administered to patients in need of such treatment. It isspecifically understood that particular subjects may, in fact, be“refractory” to a “therapeutically effective amount.” To give but oneexample, a refractory subject may have a low bioavailability such thatclinical efficacy is not obtainable. In some embodiments, reference to atherapeutically effective amount may be a reference to an amount asmeasured in one or more specific tissues (e.g., a tissue affected by thedisease, disorder or condition) or fluids (e.g., blood, saliva, serum,sweat, tears, urine, etc). Those of ordinary skill in the art willappreciate that, in some embodiments, a therapeutically effective amountmay be formulated and/or administered in a single dose. In someembodiments, a therapeutically effective amount may be formulated and/oradministered in a plurality of doses, for example, as part of a dosingregimen.

The term “treatment” (also “treat” or “treating”), in its broadestsense, refers to any administration of a substance (e.g., providedcompositions) that partially or completely alleviates, ameliorates,relives, inhibits, delays onset of, reduces severity of, and/or reducesincidence of one or more symptoms, features, and/or causes of aparticular disease, disorder, and/or condition. In some embodiments,such treatment may be administered to a subject who does not exhibitsigns of the relevant disease, disorder and/or condition and/or of asubject who exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, in some embodiments, treatmentmay be administered to a subject who exhibits one or more establishedsigns of the relevant disease, disorder and/or condition. In someembodiments, treatment may be of a subject who has been diagnosed assuffering from the relevant disease, disorder, and/or condition. In someembodiments, treatment may be of a subject known to have one or moresusceptibility factors that are statistically correlated with increasedrisk of development of the relevant disease, disorder, and/or condition.

As used herein, the term “van der Waals interaction” refers to theattractive or repulsive forces between atoms that are not due tocovalent bonds, electrostatic interactions, or hydrogen bonding.

The term “variant” refers to an entity that shows significant structuralidentity with a reference entity but differs structurally from thereference entity in the presence or level of one or more chemicalmoieties as compared with the reference entity. In many embodiments, avariant also differs functionally from its reference entity. In general,whether a particular entity is properly considered to be a “variant” ofa reference entity is based on its degree of structural identity withthe reference entity. As will be appreciated by those skilled in theart, any biological or chemical reference entity has certaincharacteristic structural elements. A variant, by definition, is adistinct chemical entity that shares one or more such characteristicstructural elements. To give but a few examples, a small molecule mayhave a characteristic core structural element (e.g., a macrocycle core)and/or one or more characteristic pendent moieties so that a variant ofthe small molecule is one that shares the core structural element andthe characteristic pendent moieties but differs in other pendentmoieties and/or in types of bonds present (single vs double, E vs Z,etc) within the core, a polypeptide may have a characteristic sequenceelement comprised of a plurality of amino acids having designatedpositions relative to one another in linear or three-dimensional spaceand/or contributing to a particular biological function, a nucleic acidmay have a characteristic sequence element comprised of a plurality ofnucleotide residues having designated positions relative to on anotherin linear or three-dimensional space. For example, a variant polypeptidemay differ from a reference polypeptide as a result of one or moredifferences in amino acid sequence and/or one or more differences inchemical moieties (e.g., carbohydrates, lipids, etc) covalently attachedto the polypeptide backbone. In some embodiments, a variant polypeptideshows an overall sequence identity with a reference polypeptide that isat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 99%. Alternatively or additionally, in some embodiments, avariant polypeptide does not share at least one characteristic sequenceelement with a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a variant polypeptide shares one or more of the biological activities ofthe reference polypeptide. In some embodiments, a variant polypeptidelacks one or more of the biological activities of the referencepolypeptide. In some embodiments, a variant polypeptide shows a reducedlevel of one or more biological activities as compared with thereference polypeptide. In many embodiments, a polypeptide of interest isconsidered to be a “variant” of a parent or reference polypeptide if thepolypeptide of interest has an amino acid sequence that is identical tothat of the parent but for a small number of sequence alterations atparticular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted ascompared with the parent. In some embodiments, a variant has 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2,or 1) number of substituted functional residues (i.e., residues thatparticipate in a particular biological activity). Furthermore, a varianttypically has not more than 5, 4, 3, 2, or 1 additions or deletions, andoften has no additions or deletions, as compared with the parent.Moreover, any additions or deletions are typically fewer than about 25,about 20, about 19, about 18, about 17, about 16, about 15, about 14,about 13, about 10, about 9, about 8, about 7, about 6, and commonly arefewer than about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent or reference polypeptide is one found in nature.As will be understood by those of ordinary skill in the art, a pluralityof variants of a particular polypeptide of interest may commonly befound in nature.

The term “wild-type” refers to an entity having a structure and/oractivity as found in nature in a “normal” (as contrasted with mutant,diseased, altered, etc) state or context. Those of ordinary skill in theart will appreciate that wild-type genes and polypeptides often exist inmultiple different forms (e.g., alleles).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Small molecules are limited in their targeting ability because theirinteractions with the target are driven by adhesive forces, the strengthof which is roughly proportional to contact surface area. Because oftheir small size, the only way for a small molecule to build up enoughintermolecular contact surface area to effectively interact with atarget protein is to be literally engulfed by that protein. Indeed, alarge body of both experimental and computational data supports the viewthat only those proteins having a hydrophobic “pocket” on their surfaceare capable of binding small molecules. In those cases, binding isenabled by engulfment.

Nature has evolved a strategy that allows a small molecule to interactwith target proteins at sites other than hydrophobic pockets. Thisstrategy is exemplified by naturally occurring immunosuppressive drugscyclosporine A, rapamycin, and FK506. The biological activity of thesedrugs involves the formation of a high-affinity complex of the smallmolecule with a small presenting protein. The composite surface of thesmall molecule and the presenting protein engages the target. Thus, forexample, the binary complex formed between cyclosporin A and cyclophilinA targets calcineurin with high affinity and specificity, but neithercyclosporin A or cyclophilin A alone binds calcineurin with measurableaffinity.

Many important therapeutic targets exert their function by complexationwith other proteins. The protein/protein interaction surfaces in many ofthese systems contain an inner core of hydrophobic side chainssurrounded by a wide ring of polar residues. The hydrophobic residuescontribute nearly all of the energetically favorable contacts, and hencethis cluster has been designated as a “hotspot” for engagement inprotein-protein interactions. Importantly, in the aforementionedcomplexes of naturally occurring small molecules with small presentingproteins, the small molecule provides a cluster of hydrophobicfunctionality akin to a hotspot, and the protein provides the ring ofmostly polar residues. In other words, presented small molecule systemsmimic the surface architecture employed widely in naturalprotein/protein interaction systems.

Nature has demonstrated the ability to reprogram the target specificityof presented small molecules—portable hotspots—through evolutionarydiversification. In the best characterized example, the complex formedbetween FK506 binding protein (FKBP) and FK506 targets calcineurin.However, FKBP can also form a complex with the related moleculerapamycin, and that complex interacts with a completely differenttarget, TorC1. To date, no methodology has been developed to reprogramthe binding and modulating ability of presenter protein/ligandinterfaces so that they can interact with and modulate other targetproteins that have previously been considered undruggable.

In addition, it is well established that some drug candidates failbecause they modulate the activity of both the intended target and othernon-intended proteins as well. The problem is particularly daunting whenthe drug binding site of the target protein is similar to binding sitesin non-target proteins. The insulin like growth factor receptor(IGF-1R), whose ATP binding pocket is structurally similar to thebinding pocket of the non-target insulin receptor (IR), is one suchexample. Small molecule development candidates that were designed totarget IGF-1R typically have the unacceptable side effect of alsomodulating the insulin receptor. However, structural dissimilarities doexist between these two proteins in the regions surrounding the ATPbinding pocket. Despite such knowledge, no methodology exists to date totake advantage of those differences and develop drugs that are specificto IGF-1R over IR.

The present invention features compounds (e.g., macrocyclic compounds)capable of modulating biological processes, for example through bindingto a presenter protein (e.g., a member of the FKBP family, a member ofthe cyclophilin family, or PIN1) and CEP250. In some embodiments, thepresenter protein is an intracellular protein. In some embodiments, thepresenter proteins is a mammalian protein. In some embodiments, providedcompounds participate in tripartite presenter protein/compound/CEP250complexes inside cells, e.g., mammalian cells. In some embodiments,provided compounds may be useful in the treatment of diseases anddisorders such as cancer, inflammation, or infections.

Compounds

The invention features compounds (e.g., macrocyclic compounds) capableof modulating biological processes, for example through binding to apresenter protein (e.g., a member of the FKBP family, a member of thecyclophilin family, or PIN1) and a target protein. In brief, thesecompounds bind endogenous intracellular presenter proteins, such as theFKBPs and the resulting binary complexes selectively bind and modulatethe activity of intracellular target proteins. Without wishing to bebound by any particular theory, we proposed that formation of atripartite complex among the presenter protein, the compound, and thetarget protein is driven by both protein-compound and protein-proteininteractions, and both are required for modulation (e.g., positive ornegative modulation) of the target protein's activity. In someembodiments, the compounds of the invention “re-program” the binding ofthe presenter proteins to protein targets that either do not normallybind to the presenter protein or have binding that is greatly enhancedin the presence of the compound thereby resulting in the ability tomodulate (e.g., positively or negatively modulate) the activity of thesenew targets.

As described herein, compounds of the invention include a presenterprotein binding moiety and a target protein interacting moiety (e.g., aCEP250 interacting moiety). In some embodiments, the presenter proteinbinding moiety and the target protein interacting moiety are separateportions of the ring structure, e.g., they do not overlap. In someembodiments, the presenter protein binding moiety and the target proteininteracting moiety are connected to one another by linkers on one orboth sides.

In some embodiments, compounds of the invention do not substantiallybind to the target protein in the absence of forming a complex, asdescribed herein. In some embodiments, a complex of a compound of theinvention and a presenter protein binds to the target protein with atleast 5-fold (at least 10-fold, at least 20-fold, at least 30-fold, atleast 40-fold, at least 50-fold, or at least 100-fold) greater affinitythan the affinity of the compound to the target protein in the absenceof forming a complex, as described herein. In certain embodiments,compounds of the invention do not substantially modulate the activity ofthe target protein in the absence forming a complex with a presenterprotein. For example, in some embodiments, the compounds of theinvention inhibit the activity of the target protein with an IC₅₀ ofgreater than 10 μM (e.g., greater than 20 μM, greater than 50 μM,greater than 100 μM, or greater than 500 μM). Alternatively, compoundsof the invention enhance the activity of the target protein with an AC₅₀of greater than 10 μM (e.g., greater than 20 μM, greater than 50 μM,greater than 100 μM, greater than 500 μM). In certain embodiments, acomplex of the compound and a presenter protein is at least 5-foldactive (i.e., has a 5-fold lower IC₅₀ or AC₅₀) than the compound alone.

Compounds (e.g., macrocyclic compounds) of the invention generally bindstrongly to the presenter protein. For example, in some embodiments, thecompounds (e.g., macrocyclic compounds) of the invention bind to thepresenter protein with a K_(D) of less than 10 μM (e.g., less than 5 μM,less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM,less than 75 nM, less than 50 nM, less than 25 nM, or less than 10 nM)or inhibit the peptidyl-prolyl isomerase activity of the presenterprotein, for example, with an IC₅₀ of less than 1 μM (e.g., less than0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM).

In some embodiments, the invention includes compounds having a structureaccording to formulae XVI to XXVI as described herein:

In certain embodiments, a compound has the structure of any of thecompounds in Table 1.

In some embodiments, a compound is a natural compound (e.g., synthesizedby a genetically unmodified bacterial strain). In some embodiments, acompound is a variant of a natural compound (e.g., a semi-syntheticcompound). In some embodiments, a variant shares ring size with thereference natural compound. In some embodiments, a variant differs fromthe reference natural compound only by identity of one or moresubstituents (e.g., for at least one position, the variant has adifferent substitutent or set of substituents than is found at thecorresponding position in an appropriate reference compound).

In some embodiments, compounds of the invention (e.g., macrocycliccompounds of the invention) include 12 to 40 ring atoms (e.g., 12 to 20ring atoms, 14 to 20 ring atoms, 17 to 25 ring atom, 21 to 26 ringatoms, 20 to 30 ring atoms, 25 to 35 ring atoms, 30 to 40 ring atoms).In some embodiments, such compounds include 19 ring atoms. In someembodiments, such compounds have an even number of ring atoms. Incertain embodiments, at least 25% (e.g., at least 30%, at least 35%, atleast 40%, at least 45%) of the atoms in the compound are included in asingle or fused ring system.

In some embodiments, compounds of the invention include a ring (e.g., amacrocycle) whose ring atoms are selected from the group consisting ofcarbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, sulfuratoms, phosphorous atoms, silicon atoms and combinations thereof; insome embodiments all ring atoms in the compound are selected from thisgroup. In some embodiments, compounds of the invention include a ring(e.g., a macrocycle) whose ring atoms are selected only from the groupconsisting carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atomsand combinations thereof; in some embodiments, all ring atoms in thecompound are selected only from the group consisting carbon atoms,hydrogen atoms, nitrogen atoms, oxygen atoms and combinations thereof.

In certain embodiments, ring linkage in provided compounds (e.g.,macrocyclic compounds) of the invention includes a ketone, an ester, anamide, an ether, a thioester, a urea, an amidine, or a hydrocarbon.

In some embodiments, a provided compound is non-peptidal. In certainembodiments, a provided compound includes one or more amino acidresidues. In some embodiments, a provided compound includes only aminoacid residues.

In some embodiments, the molecular weight of compounds of the inventionis between 400 and 2000 daltons (e.g., 400 to 600 daltons, 500 to 700daltons, 600 to 800 daltons, 700 to 900 daltons, 800 to 1000 daltons,900 to 1100 daltons, 1000 to 1200 daltons, 1100 to 1300 daltons, 1200 to1400 daltons, 1300 to 1500 daltons, 1400 to 1600 daltons, 1500 to 1700daltons, 1600 to 1800 daltons, 1700 to 1900 daltons, 1800 to 2000daltons, 400 to 1000 daltons, 1000-2000 daltons). In some embodiments,the molecular weight of compounds of the invention is less than 2000daltons (e.g., less than 500 daltons, less than 600 daltons, less than700 daltons, less than 800 daltons, less than 900 daltons, less than1000 daltons, less than 1100 daltons, less than 1200 daltons, less than1300 daltons, less than 1400 daltons, less than 1500 daltons, less than1600 daltons, less than 1700 daltons, less than 1800 daltons, less than1900 daltons).

In certain embodiments, molecule provided compound is hydrophobic. Forexample, in some embodiments, compounds have a c Log P of equal to orgreater than 2 (e.g., equal to or greater than 2.5, equal to or greaterthan 3.0, equal to or greater than 3.5, equal to or greater than 4,equal to or greater than 4.5, equal to or greater than 5, equal to orgreater than 5.5, equal to or greater than 6, equal to or greater than6.5, equal to or greater than 7). Alternatively, in some embodiments,compounds have a c Log P of between 2 and 7 (e.g., between 2 and 4,between 3.5 and 4.5, between 4 and 5, between 4.5 and 5.5, between 5 and6, between 5.5 and 6.5, between 6 and 7, between 4 and 7, between 4 and6, between 4 and 5.5). A provided compound may also be characterized ashydrophobic by having low solubility in water. For example, in someembodiments, compounds have a solubility of greater than 1 μM in water(e.g., greater than 1 μM, greater than 2 μM, greater than 5 μM, greaterthan 10 μM, greater than 20 μM, greater than 30 μM, greater than 40 μM,greater than 50 μM, greater than 75 μM, greater than 100 μM).Alternatively, in some embodiments, compounds have a solubility in waterof between 1-100 μM (e.g., 1-10 μM, 5-10 μM, 5-20 μM, 10-50 μM, 5-50 μM,20-100 μM).

In some embodiments, compounds of the invention are cell penetrant(e.g., they are able to enter the intracellular domain of a cell withoutkilling the cell and/or are capable of entering the intercellular domainwhen contacted with extracellular environs).

Compounds of the invention may or may not be naturally occurring. Insome embodiments, compounds of the invention are not naturallyoccurring. In certain embodiments, compounds of the invention areengineered. An engineered compound is a compound whose design and/orproduction involves action of the hand of man (e.g., a compound preparedby chemical synthesis, a compound prepared by a cell that has beengenetically manipulated relative to a reference wild type cell, acompound produced by a cell in culture conditions modified to enhanceproduction of the compound).

Presenter Protein Binding Moiety

Compounds of the invention include a presenter protein binding moiety.This moiety includes the group of ring atoms (e.g., 5 to 20 ring atoms,5 to 10 ring atoms, 10 to 20 ring atoms) and the moieties attachedthereto (e.g., atoms within 20 atoms of a ring atom such as, atomswithin 15 atoms of a ring atom, atoms within 10 atoms of a ring atom,atoms within 5 atoms of a ring atom) that participate in binding to apresenter protein such that a provided compound specifically binds tosaid presenter protein, for example, with a K_(D) of less than 10 μM(e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM,less than 10 nM) or inhibits the peptidyl-prolyl isomerase activity ofthe presenter protein, for example, with an IC₅₀ of less than 1 μM(e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than0.01 μM). In some embodiments, the presenter protein binding moiety doesnot encompass the entirety of atoms in a provided compound that interactwith the presenter protein. In some embodiments, one or more atoms ofthe presenter protein binding moiety may be within the CEP250interacting moiety. In certain embodiments, one or more atoms of thepresenter protein binding moiety do not interact with the presenterprotein.

In some embodiments, a presenter protein binding moiety includes aN-acyl proline moiety, a N-acyl-pipecolic acid moiety, a N-acyl3-morpholino-carboxylic acid moiety, and/or a N-acyl piperzic acidmoiety (e.g., with acylation on either nitrogen atom. In certainembodiments, a presenter protein binding moiety includes aN-acyl-pipecolic acid moiety. In some embodiments, a presenter proteinbinding moiety includes a N-acyl proline moiety. In certain embodiments,a presenter protein binding moiety includes a N-acyl3-morpholino-carboxylic acid moiety. In some embodiments, a presenterprotein binding moiety includes a N-acyl piperzic acid moiety.

In some embodiments, at least one atom of a presenter protein bindingmoiety participates in binding with one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,or fifteen) of Tyr 27, Phe 37, Asp 38, Arg 41, Phe 47, Gln 54, Glu 55,Val 56, Ile 57, Trp 60, Ala 82, Try 83, His 88, Ile 92, and/or Phe 100of FKBP12. In some embodiments, at least one at of a presenter proteinbinding moiety participates in binding with at least one (e.g., two,three, or four) of Arg 41, Gln 54, Glu 55, and/or Ala 82 of FKBP12.

In some embodiments, a presenter protein binding moiety has a structureaccording to Formula I-VIII:

In some embodiments, a presenter protein binding moiety has a structureaccording to Formula Ia-IVa:

In some embodiments, a presenter protein binding moiety includes orconsists of the structure:

or a stereo isomer thereof.

In certain embodiments, the presenter protein binding moiety is orincludes the structure:

Target Protein Interacting Moiety

Compounds of the invention include a target protein interacting moiety(e.g., a CEP250 interacting moiety). This moiety includes the group ofring atoms (e.g., 5 to 20 ring atoms, 5 to 10 ring atoms, 10 to 20 ringatoms) and the moieties attached thereto (e.g., atoms within 20 atoms ofa ring atom such as, atoms within 15 atoms of a ring atom, atoms within10 atoms of a ring atom, atoms within 5 atoms of a ring atom) that whenthe compound is in a complex with a presenter protein, specifically bindto the target protein. In some embodiments, a target protein interactingmoiety comprises a plurality of the atoms in the compound that interactwith the target protein. In some embodiments, one or more atoms of atarget protein interacting moiety may be within the presenter proteinbinding moiety. In certain embodiments, one or more atoms of a targetprotein interacting moiety do not interact with the target protein.

The target protein can bind to a ring atom in a target proteininteracting moiety. Alternatively, the target protein can bind to two ormore ring atoms in a target protein interacting moiety. In anotheralternative, the target protein can bind to a substituent attached toone or more ring atoms in a target protein interacting moiety. Inanother alternative, the target protein can bind to a ring atom in atarget protein interacting moiety and to a substituent attached to oneor more ring atoms in a target protein interacting moiety (e.g., aCEP250 interacting moiety). In another alternative, the target proteinbinds to a group that mimics a natural ligand of the target protein andwherein the group that mimics a natural ligand of the target protein isattached to a target protein interacting moiety (e.g., a CEP250interacting moiety). In yet another alternative, the target proteinbinds to a presenter protein and the affinity of the target protein fora presenter protein in the binary complex is increased relative to theaffinity of the target protein for a presenter protein in the absence ofthe complex. Binding in these examples is typically through, but notlimited to non-covalent interactions of the target protein to a targetprotein interacting moiety (e.g., a CEP250 interacting moiety).

In some embodiments, a target protein interacting moiety (e.g., a CEP250interacting moiety) is hydrophobic. For example, in some embodiments, atarget protein interacting moiety (e.g., a CEP250 interacting moiety)has a c Log P of equal to or greater than 2 (e.g., equal to or greaterthan 2.5, equal to or greater than 3, equal to or greater than 3.5,equal to or greater than 4, equal to or greater than 4.5, equal to orgreater than 5, equal to or greater than 5.5, equal to or greater than6, equal to or greater than 6.5, or equal to or greater than 7).Alternatively, in some embodiments, a target protein interacting moiety(e.g., a CEP250 interacting moiety) has a c Log P of between 2 and 7(e.g., between 2 and 4, between 2.5 and 4.5, between 3 and 5, between3.5 and 5.5, between 4 and 6, between 4.5 and 6.5, between 5 and 7,between 3 and 6, between 3 and 5, or between 3 and 5.5). A targetprotein interacting moiety (e.g., a CEP250 interacting moiety) may alsobe characterized as hydrophobic by having low polar surface area. Forexample, in some embodiments, a target protein interacting moiety (e.g.,a CEP250 interacting moiety) has a polar surface area of less than 350Å² (e.g., less than 300 Å², less than 250 Å², less than 200 Å², lessthan 150 Å², or less than 125 Å²).

In some embodiments, a target protein interacting moiety (e.g., a CEP250interacting moiety) comprises one or more hydrophobic pendant groups(e.g., one or more methyl, ethyl, isopropyl, phenyl, benzyl, and/orphenethyl groups). In some embodiments, the pendant groups comprisefewer than 30 total atoms (e.g., fewer than 25 total atoms, fewer than20 total atoms, fewer than 15 total atoms, or fewer than 10 totalatoms.) Alternatively, in some embodiments, the pendant groups comprisebetween 10 and 30 total atoms (e.g., 10 to 20 total atoms, 15 to 25total atoms, or 20 to 30 total atoms). In certain embodiments thependant groups have a molecular weight less than 200 daltons (e.g., lessthan 150 daltons, less than 100 daltons, less than 75 daltons, or lessthan 50 daltons). Alternatively, in some embodiments, the pendant groupshave a molecular weight between 50 to 200 daltons (e.g., 50 to 100daltons, 75 to 150 daltons, or 100 to 200 daltons).

In some embodiments, a target protein interacting moiety (e.g., a CEP250interacting moiety) is hydrocarbon based (e.g., the moiety comprisesmostly carbon-carbon bonds). In some embodiments, a target proteininteracting moiety (e.g., a CEP250 interacting moiety) is hydrocarbonbased and includes a linear bivalent C₄-C₃₀ (e.g., C₆-C₂₀ or C₆-C₁₅)aliphatic group consisting predominantly of carbon and hydrogen,optionally including one or more double bonds. In some embodiments, thebivalent aliphatic group can also be substituted with a group thatmimics a natural ligand that binds to the target protein. Examplesinclude phosphotyrosine mimics and ATP mimetics.

In some embodiments, a target protein interacting moiety (e.g., a CEP250interacting moiety) is peptide based (e.g., the moiety comprises peptidebonds). In some embodiments, a target protein interacting moiety (e.g.,a CEP250 interacting moiety) is peptide based and includes one or more(e.g., two, three, four, five, six, seven, or eight) alanine residues,one or more (e.g., two, three, four, five, six, seven, or eight) valineresidues, one or more isoleucine (e.g., two, three, four, five, six,seven, or eight) residues, one or more leucine (e.g., two, three, four,five, six, seven, or eight) residues, one or more methionine (e.g., two,three, four, five, six, seven, or eight) residues, one or morephenylalanine (e.g., two, three, four, five, six, seven, or eight)residues, one or more (e.g., two, three, four, five, six, seven, oreight) tyrosine residues, one or more (e.g., two, three, four, five,six, seven, or eight) tryptophan residues, one or more (e.g., two,three, four, five, six, seven, or eight) glycine residues, and/or one ormore (e.g., two, three, four, five, six, seven, or eight) prolineresidues. In some embodiments, a target protein interacting moiety(e.g., a CEP250 interacting moiety) is peptide based and includes one ormore (e.g., two, three, four, five, six, seven, or eight) arginineresidues or one or more (e.g., two, three, four, five, six, seven, oreight) lysine residues. In some embodiments, a target proteininteracting moiety (e.g., a CEP250 interacting moiety) is peptide basedand includes one or more (e.g., two, three, four, five, six, seven, oreight) non-natural amino acids, one or more (e.g., two, three, four,five, six, seven, or eight) D-amino acids, and/or one or more (e.g.,two, three, four, five, six, seven, or eight)N-alkylated amino acids. Insome embodiments, a target protein interacting moiety (e.g., a CEP250interacting moiety) is peptide based and includes predominantly D-aminoacids (e.g., at least 50% of the amino acids are D-amino acids, at least75% of the amino acids are D-amino acids, 100% of the amino acids areD-amino acids). In certain embodiments, a target protein interactingmoiety (e.g., a CEP250 interacting moiety) is peptide based and includespredominantly N-alkylated amino acids (e.g., at least 50% of the aminoacids are N-alkylated amino acids, at least 75% of the amino acids areN-alkylated amino acids, 100% of the amino acids are N-alkylated aminoacids). In certain embodiments, a target protein interacting moiety(e.g., a CEP250 interacting moiety) is peptide based and includes one ormore (e.g., two, three, four, five, six, seven, or eight)depsi-linkages.

In some embodiments, the target protein interacting moiety (e.g., CEP250interacting moiety) has the structure:

Linkers

The compounds of the invention include a linker (e.g., two linkersconnecting the presenter protein binding moiety and target proteininteracting moiety). The linker component of the invention is, at itssimplest, a bond, but may also provide a linear, cyclic, or branchedmolecular skeleton having pendant groups covalently linking twomoieties.

In some embodiments, at least one atom of a linker participates inbinding to the presenter protein and/or the target protein. In certainembodiments, at least one atom of a linker does not participate inbinding to the presenter protein and/or the target protein.

Thus, linking of the two moieties is achieved by covalent means,involving bond formation with one or more functional groups located oneither moiety. Examples of chemically reactive functional groups whichmay be employed for this purpose include, without limitation, amino,hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinaldiols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl,imidazolyl, and phenolic groups.

The covalent linking of the two moieties may be effected using a linkerthat contains reactive moieties capable of reaction with such functionalgroups present in either moiety. For example, an amine group of a moietymay react with a carboxyl group of the linker, or an activatedderivative thereof, resulting in the formation of an amide linking thetwo.

Examples of moieties capable of reaction with sulfhydryl groups includeα-haloacetyl compounds of the type XCH₂CO— (where X=Br, Cl, or I), whichshow particular reactivity for sulfhydryl groups, but which can also beused to modify imidazolyl, thioether, phenol, and amino groups asdescribed by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimidederivatives are also considered selective towards sulfhydryl groups, butmay additionally be useful in coupling to amino groups under certainconditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry12:3266 (1973)), which introduce a thiol group through conversion of anamino group, may be considered as sulfhydryl reagents if linking occursthrough the formation of disulfide bridges.

Examples of reactive moieties capable of reaction with amino groupsinclude, for example, alkylating and acylating agents. Representativealkylating agents include:

(i) α-haloacetyl compounds, which show specificity towards amino groupsin the absence of reactive thiol groups and are of the type XCH₂CO—(where X=Br, Cl, or I), for example, as described by Wong Biochemistry24:5337 (1979);

(ii) N-maleimide derivatives, which may react with amino groups eitherthrough a Michael type reaction or through acylation by addition to thering carbonyl group, for example, as described by Smyth et al., J. Am.Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964);

(iii) aryl halides such as reactive nitrohaloaromatic compounds;

(iv) alkyl halides, as described, for example, by McKenzie et al., J.Protein Chem. 7:581 (1988);

(v) aldehydes and ketones capable of Schiff's base formation with aminogroups, the adducts formed usually being stabilized through reduction togive a stable amine;

(vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, whichmay react with amino, sulfhydryl, or phenolic hydroxyl groups;

(vii) chlorine-containing derivatives of s-triazines, which are veryreactive towards nucleophiles such as amino, sufhydryl, and hydroxylgroups;

(viii) aziridines based on s-triazine compounds detailed above, e.g., asdescribed by Ross, J. Adv. Cancer Res. 2:1 (1954), which react withnucleophiles such as amino groups by ring opening;

(ix) squaric acid diethyl esters as described by Tietze, Chem. Ber.124:1215 (1991); and

(x) α-haloalkyl ethers, which are more reactive alkylating agents thannormal alkyl halides because of the activation caused by the etheroxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463(1993).

Representative amino-reactive acylating agents include:

(i) isocyanates and isothiocyanates, particularly aromatic derivatives,which form stable urea and thiourea derivatives respectively;

(ii) sulfonyl chlorides, which have been described by Herzig et al.,Biopolymers 2:349 (1964);

(iii) acid halides;

(iv) active esters such as nitrophenylesters or N-hydroxysuccinimidylesters;

(v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides;

(vi) other useful reagents for amide bond formation, for example, asdescribed by M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag, 1984;

(vii) acylazides, e.g., wherein the azide group is generated from apreformed hydrazide derivative using sodium nitrite, as described byWetz et al., Anal. Biochem. 58:347 (1974);

(viii) imidoesters, which form stable amidines on reaction with aminogroups, for example, as described by Hunter and Ludwig, J. Am. Chem.Soc. 84:3491 (1962); and

(ix) haloheteroaryl groups such as halopyridine or halopyrimidine.

Aldehydes and ketones may be reacted with amines to form Schiff's bases,which may advantageously be stabilized through reductive amination.Alkoxylamino moieties readily react with ketones and aldehydes toproduce stable alkoxamines, for example, as described by Webb et al., inBioconjugate Chem. 1:96 (1990).

Examples of reactive moieties capable of reaction with carboxyl groupsinclude diazo compounds such as diazoacetate esters and diazoacetamides,which react with high specificity to generate ester groups, for example,as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxylmodifying reagents such as carbodiimides, which react through O-acylureaformation followed by amide bond formation, may also be employed.

It will be appreciated that functional groups in either moiety may, ifdesired, be converted to other functional groups prior to reaction, forexample, to confer additional reactivity or selectivity. Examples ofmethods useful for this purpose include conversion of amines tocarboxyls using reagents such as dicarboxylic anhydrides; conversion ofamines to thiols using reagents such as N-acetylhomocysteinethiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, orthiol-containing succinimidyl derivatives; conversion of thiols tocarboxyls using reagents such as α-haloacetates; conversion of thiols toamines using reagents such as ethylenimine or 2-bromoethylamine;conversion of carboxyls to amines using reagents such as carbodiimidesfollowed by diamines; and conversion of alcohols to thiols usingreagents such as tosyl chloride followed by transesterification withthioacetate and hydrolysis to the thiol with sodium acetate.

So-called zero-length linkers, involving direct covalent joining of areactive chemical group of one moiety with a reactive chemical group ofthe other without introducing additional linking material may, ifdesired, be used in accordance with the invention.

More commonly, however, the linker will include two or more reactivemoieties, as described above, connected by a spacer element. Thepresence of such a spacer permits bifunctional linkers to react withspecific functional groups within either moiety, resulting in a covalentlinkage between the two. The reactive moieties in a linker may be thesame (homobifunctional linker) or different (heterobifunctional linker,or, where several dissimilar reactive moieties are present,heteromultifunctional linker), providing a diversity of potentialreagents that may bring about covalent attachment between the twomoieties.

Spacer elements in the linker typically consist of linear or branchedchains and may include a C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₂₋₆heterocyclyl, C₆₋₁₂ aryl, C₇₋₁₄ alkaryl, C₃₋₁₀ alkheterocyclyl, C₂-C₁₀₀polyethylene glycol, or C₁₋₁₀ heteroalkyl.

In some instances, the linker is described by Formula V.

Examples of homobifunctional linkers useful in the preparation ofconjugates of the invention include, without limitation, diamines anddiols selected from ethylenediamine, propylenediamine andhexamethylenediamine, ethylene glycol, diethylene glycol, propyleneglycol, 1,4-butanediol, 1,6 hexanediol, cyclohexanediol, andpolycaprolactone diol.

In some embodiments, the linker is a bond or a linear chain of up to 10atoms, independently selected from carbon, nitrogen, oxygen, sulfur orphosphorous atoms, wherein each atom in the chain is optionallysubstituted with one or more substituents independently selected fromalkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro,hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino,acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and wherein anytwo atoms in the chain may be taken together with the substituents boundthereto to form a ring, wherein the ring may be further substitutedand/or fused to one or more optionally substituted carbocyclic,heterocyclic, aryl, or heteroaryl rings.

In some embodiments, the linker has the structure of Formula XXIX:

A¹-(B¹)_(a)—(C¹)_(b)—(B²)_(c)-(D)-(B³)_(d)—(C²)_(e)—(B⁴)_(f)-A²  Formula XXIX

where A¹ is a bond between the linker and presenter protein bindingmoiety; A² is a bond between the mammalian target interacting moiety andthe linker; B¹, B², B³, and B⁴ each, independently, is selected fromoptionally substituted C₁-C₂ alkyl, optionally substituted C₁-C₃heteroalkyl, O, S, and NR^(N); RN is hydrogen, optionally substitutedC₁₋₄ alkyl, optionally substituted C₃₋₄ alkenyl, optionally substitutedC₂₋₄ alkynyl, optionally substituted C₂₋₆ heterocyclyl, optionallysubstituted C₆₋₁₂ aryl, or optionally substituted C₁₋₇ heteroalkyl; C₁and C₂ are each, independently, selected from carbonyl, thiocarbonyl,sulphonyl, or phosphoryl; a, b, c, d, e, and f are each, independently,0 or 1; and D is optionally substituted C₁₋₁₀ alkyl, optionallysubstituted C₂₋₁₀ alkenyl, optionally substituted C₂₋₁₀ alkynyl,optionally substituted C₂₋₆ heterocyclyl, optionally substituted C₆₋₁₂aryl, optionally substituted C₂-C₁₀₀ polyethylene glycol, or optionallysubstituted C₁₋₁₀ heteroalkyl, or a chemical bond linkingA¹-(B¹)_(a)—(C¹)_(b)—(B²)_(c)— to —(B³)_(d)—(C²)_(e)—(B⁴)_(f)-A².

Compound Characteristics

Pharmacokinetic Parameters

Preliminary exposure characteristics of the compounds can be evaluatedusing, e.g., an in vivo Rat Early Pharmacokinetic (EPK) study design toshow bioavailability. For example, Male Sprague-Dawley rats can be dosedvia oral (PO) gavage in a particular formulation. Blood samples can thenbe collected from the animals at 6 timepoints out to 4 hours post-dose.Pharmacokinetic analysis can then performed on the LC-MS/MS measuredconcentrations for each timepoint of each compound.

Cell Permeability

In some embodiments, the compound is cell penetrant. To determinepermeability of a compound any method known in the art may be employedsuch as a Biosensor assay as described herein.

Presenter Proteins

Presenter proteins can bind a small molecule to form a complex, whichcan bind to and modulate the activity of a target protein (e.g., aeukaryotic target protein such as a mammalian target protein or a fungaltarget protein or a prokaryotic target protein such as a bacterialtarget protein). In some embodiments, the presenter protein is amammalian presenter protein (e.g., a human presenter protein). In someembodiments, the presenter protein is a fungal presenter protein. Incertain embodiments, the presenter protein is a bacterial presenterprotein. In some embodiments, the presenter protein is a plant presenterprotein. In some embodiments, the presenter protein is a relativelyabundant protein (e.g., the presenter protein is sufficiently abundantthat participation in a tripartite complex does not materiallynegatively impact the biological role of the presenter protein in a celland/or viability or other attributes of the cell). In some embodiments,the presenter protein is more abundant than the target protein. Incertain embodiments, the presenter protein is a protein that haschaperone activity within a cell. In some embodiments, the presenterprotein has multiple natural interaction partners within a cell. Incertain embodiments, the presenter protein is one which is known to binda small molecule to form a binary complex that is known to or suspectedof binding to and modulating the biological activity of a targetprotein. Immunophilins are a class of presenter proteins which are knownto have these functions and include FKBPs and cyclophilins. In someembodiments, a reference presenter protein exhibits peptidyl prolylisomerase activity; in some embodiments, a presenter protein showscomparable activity to the reference presenter protein. In certainembodiments, the presenter protein is a member of the FKBP family (e.g.,FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36,FKBP38, FKBP51, FKBP52, FKBP60, FKBP65, and FKBP133), a member of thecyclophilin family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR,SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1,PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, or PPIAL4G), or PIN1. The “FKBPfamily” is a family of proteins that have prolyl isomerase activity andfunction as protein folding chaperones for proteins containing prolineresidues. Genes that encode proteins in this family include AIP, AIPL1,FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9,FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and LOC541473.

The “cyclophilin family” is a family of proteins that bind tocyclosporine. Genes that encode proteins in this family include PPIA,PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1, PPIL2,PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary cyclophilins includeCyp-A, PPIL1, PPIL3, USA-Cyp, Cyp-F, Cyp-B, Cyp-C, Cyp29, Cyp33, Cyp40,SDCCAG10, Cyp57, Cyp60, HAL539, Cyp88, NK-Cyp and RanBP2.

In some embodiments, a presenter protein is a chaperone protein such asGRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, Proteindisulfide isomerase (PDI), and ERp57.

In some embodiments, a presenter protein is an allelic variant or splicevariant of a FKBP or cyclophilindisclosed herein.

In some embodiments, a presenter protein is a polypeptide whose aminoacid sequence i) shows significant identity with that of a referencepresenter protein; ii) includes a portion that shows significantidentity with a corresponding portion of a reference presenter protein;and/or iii) includes at least one characteristic sequence found inpresenter protein. In many embodiments, identity is considered“significant” for the purposes of defining an presenter protein if it isabove 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher. In some 170, 180, 190, 200,210, 220, 230, 240, 250, 300, 350, 450, 500, 550, or 600 amino acids ormore.

Representative presenter proteins are encoded by the genes or homologsthereof listed in Table 3; in some embodiments, a reference presenterprotein is encoded by a gene set forth in Table 2. Also, those ofordinary skill in the art, referring to Table 2, can readily identifysequences that are characteristic of presenter proteins generally,and/or of particular subsets of presenter proteins.

TABLE 2 Genes that Encode Selected Presenter Proteins Gene Name UniprotAccession Number AIP O00170 AIPL1 Q9NZN9 FKBP1A P62942 FKBP1B P68106FKBP2 P26885 FKBP3 Q00688 FKBP4 Q02790 FKBP5 Q13451 FKBP6 O75344 FKBP7Q9Y680 FKBP8 Q14318 FKBP9 O95302 FKBP9L Q75LS8 FKBP10 Q96AY3 FKBP11Q9NYL4 FKBP14 Q9NWM8 FKBP15 Q5T1M5 LOC541473 — PPIA Q567Q0 PPIB P23284PPIC P45877 PPID Q08752 PPIE Q9UNP9 PPIG Q13427 PPIH O43447 PPIL1 Q9Y3C6PPIL2 Q13356 PPIL3 Q9H2H8 PPIL4 Q8WUA2 PPIL5 Q32Q17 PPIL6 Q8IXY8 PPWD1Q96BP3

Target Proteins

A target protein (e.g., a eukaryotic target protein such as a mammaliantarget protein or a fungal target protein or a prokaryotic targetprotein such as a bacterial target protein) is a protein which mediatesa disease condition or a symptom of a disease condition. As such, adesirable therapeutic effect can be achieved by modulating (inhibitingor increasing) its activity. Target proteins useful in the complexes andmethods of the invention include those which do not naturally associatewith a presenter protein, e.g., those which have an affinity for apresenter protein in the absence of a binary complex with a compound ofthe invention of greater than 1 μM, preferably greater than 5 μM, andmore preferably greater than 10 μM. Alternatively, target proteins whichdo not naturally associate with a presenter protein are those which havean affinity for a compound of the invention in the absence of a binarycomplex greater than 1 μM, preferably greater than 5 μM, and morepreferably greater than 10 μM. In another alternative, target proteinswhich do not naturally associate with a presenter protein are thosewhich have an affinity for a binary complex of cyclosporine, rapamycin,or FK506 and a presenter protein (e.g., FKBP) of greater than 1 μM,preferably greater than 5 μM, and more preferably greater than 10 μM. Inyet another alternative, target proteins which do not naturallyassociate with a presenter protein are those which are other thancalcineurin or mTOR. The selection of suitable target proteins for thecomplexes and methods of the invention may depend on the presenterprotein. For example, target proteins that have low affinity for acyclophilin may have high affinity for an FKBP and would not be usedtogether with the latter.

Target proteins can be naturally occurring, e.g., wild type.Alternatively, a target protein can vary from the wild type protein butstill retain biological function, e.g., as an allelic variant, a splicemutant or a biologically active fragment.

In some embodiments, a target protein is a transmembrane protein. Insome embodiments, a target protein has a coiled coil structure. Incertain embodiments, a target protein is one protein of a dimericcomplex.

In some embodiments, a target protein of the invention includes one ormore surface sites (e.g., a flat surface site) characterized in that, inthe absence of forming a presenter protein/compound complex, smallmolecules typically demonstrate low or undetectable binding to thesite(s). In some embodiments, a target protein includes one or moresurface sites (e.g., a flat surface site) to which, in the absence offorming a presenter protein/compound complex, a particular smallmolecule (e.g., the compound) shows low or undetectable binding (e.g.,binding at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 fold ormore lower than that observed with a presenter protein/compound complexinvolving the same compound). In some embodiments, a target protein hasa surface characterized by one or more sites (and, in some embodiments,an entire surface) that lack(s) any a traditional binding pocket, forexample, a cavity or pocket on the protein structure with physiochemicaland/or geometric properties comparable to proteins whose activity hasbeen modulated by one or more small molecules. In certain embodiments, atarget protein has a traditional binding pocket and a site for aprotein-protein interaction. In some embodiments, a target protein is anundruggable target, for example, a target protein is not a member of aprotein family which is known to be targeted by drugs and/or does notpossess a binding site that is expected (e.g., according to art-acceptedunderstanding, as discussed herein) to be suitable for binding to asmall molecule.

In some embodiments, the target protein is CEP250.

Complexes

Presenter Protein/Compound Complexes

In naturally occurring protein-protein interactions, the binding eventis driven largely by hydrophobic residues on flat surface sites of thetwo proteins, in contrast to many small molecule-protein interactionswhich are driven by interactions between the small molecule in a cavityor pocket on the protein. The hydrophobic residues on the flat surfacesite form hydrophobic hot spots on the two interacting proteins whereinmost of the binding interactions between the two proteins are van derWaals interactions. Small molecules may be used as portable hotspots forproteins which are lacking one (e.g., presenter proteins) through theformation of complexes (e.g., a presenter protein/compound complex) toparticipate in pseudo protein-protein interactions (e.g., forming atripartite complex with a target protein).

Many mammalian proteins are able to bind to any of a plurality ofdifferent partners; in some cases, such alternative binding interactionscontribute to biological activity of the proteins. Many of theseproteins adapt the inherent variability of the hot spot protein regionsto present the same residues in different structural contexts. Morespecifically, the protein-protein interactions can be mediated by aclass of natural products produced by a select group of fungal andbacterial species. These molecules exhibit both a common structuralorganization and resultant functionality that provides the ability tomodulate protein-protein interaction. These molecules contain apresenter protein binding moiety that is highly conserved and a targetprotein interacting moiety that exhibits a high degree of variabilityamong the different natural products. The presenter protein bindingmoiety confers specificity for the presenter protein and allows themolecule to bind to the presenter protein to form a binary complex; themammalian target protein interacting moiety confers specificity for thetarget protein and allows the binary complex to bind to the targetprotein, typically modulating (e.g., positively or negativelymodulating) its activity.

These natural products are presented by presenter proteins, such asFKBPs and cyclophilins and act as diffusible, cell-penetrant, orallybio-available adaptors for protein-protein interactions. Examplesinclude well known and clinically relevant molecules such as Rapamycin(Sirolimus), FK506 (Tacrolimus), and Cyclosporin. In brief, thesemolecules bind endogenous intracellular presenter proteins, the FKBPse.g. rapamycin and FK506 or cyclophilins e.g. diluents, and theresulting binary complexes of presenter protein-bound moleculesselectively bind and inhibit the activity of intracellular targetproteins. Formation of a tripartite complex between the presenterprotein, the molecule, and the target protein is driven by bothprotein-molecule and protein-protein interactions and both are requiredfor inhibition of the target protein. In the example of theFKBP-rapamycin complex, the intracellular target is the serine-threoninekinase mTOR, whereas for FKBP-FK506 complex, the intracellular target isthe phosphatase calcineurin. Of particular interest in the preceding twoexamples, FKBP12 is utilized as a partner presentation protein by boththe rapamycin and FK506 presentation ligands. Moreover, thesub-structure components of rapamycin and FK506 responsible for bindingto FKBP12 are closely related structurally, i.e. the so-called“Conserved Region,” but it is the dramatic structural differencesbetween rapamycin and FK506 in the non FKBP12-binding regions, i.e. the“Variable Region,” that results in the specific targeting of twodistinct intracellular proteins, mTOR and calcineurin, respectively. Inthis fashion, the Variable Regions of rapamycin and FK506 are serving ascontributors to the binding energy necessary for enabling presenterprotein-target protein interaction.

In some embodiments, a presenter protein/compound complex binds to thetarget protein with at least 5-fold (e.g., at least 10-fold, at least20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or atleast 100-fold) greater affinity than the complex binds to each of mTORand/or calcineurin.

In some embodiments, a presenter protein/compound complex binds to thetarget protein with at least 5-fold (e.g., at least 10-fold, at least20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or atleast 100-fold) greater affinity than the affinity of the compound tothe target protein when the compound is not bound in a complex with apresenter protein.

In certain embodiments, a presenter protein/compound complex binds tothe target protein with at least 5-fold (e.g., at least 10-fold, atleast 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, atleast 100-fold) greater affinity than the affinity of the presenterprotein to the target protein when the presenter protein is not bound ina complex with a compound.

In some embodiments, a presenter protein/compound complex inhibits anaturally occurring interaction between the target protein and a ligand,such as a protein or a small molecule that specifically binds to thetarget protein.

In certain embodiments, when the presenter protein is a prolylisomerase, the prolyl isomerase activity is inhibited by formation ofthe presenter protein/compound complex. In some embodiments of thepresenter protein/compound complexes of the invention, the compoundspecifically binds to said presenter protein with a K_(D) of less than10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25nM, or less than 10 nM) or inhibits the peptidyl-prolyl isomeraseactivity of the presenter protein, for example, with an IC₅₀ of lessthan 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM,or less than 0.01 μM).

Tripartite Complexes

The vast majority of small molecule drugs act by binding a functionallyimportant site on a target protein, thereby modulating (e.g., positivelyor negatively modulating) the activity of that protein. For example, thecholesterol-lowering drugs statins bind the enzyme active site ofHMG-CoA reductase, thus preventing the enzyme from engaging with itssubstrates. The fact that many such drug/target interacting pairs areknown may have misled some into believing that a small moleculemodulator could be discovered for most, if not all, proteins provided areasonable amount of time, effort, and resources. This is far from thecase. Current estimates hold that only about 10% of all human proteinsare targetable by small molecules. The other 90% are currentlyconsidered refractory or intractable toward small molecule drugdiscovery. Such targets are commonly referred to as “undruggable.” Theseundruggable targets include a vast and largely untapped reservoir ofmedically important human proteins. Thus, there exists a great deal ofinterest in discovering new molecular modalities capable of modulatingthe function of such undruggable targets.

The present invention encompasses the recognition that small moleculesare typically limited in their targeting ability because theirinteractions with the target are driven by adhesive forces, the strengthof which is roughly proportional to contact surface area. Because oftheir small size, the only way for a small molecule to build up enoughintermolecular contact surface area to effectively interact with atarget protein is to be literally engulfed by that protein. Indeed, alarge body of both experimental and computational data supports the viewthat only those proteins having a hydrophobic “pocket” on their surfaceare capable of binding small molecules. In those cases, binding isenabled by engulfment. Not a single example exists of a small moleculebinding with high-affinity to a protein outside of a hydrophobic pocket.

Nature has evolved a strategy that allows a small molecule to interactwith target proteins at sites other than hydrophobic pockets. Thisstrategy is exemplified by the naturally occurring immunosuppressivedrugs cyclosporine A, rapamycin, and FK506. The activity of these drugsinvolves the formation of a high-affinity complex of the small moleculewith a small presenting protein. The composite surface of the smallmolecule and the presenting protein then engages the target. Thus, forexample, the binary complex formed between cyclosporine A andcyclophilin A targets calcineurin with high affinity and specificity,but neither cyclosporine A or cyclophilin A alone binds calcineurin withmeasurable affinity.

Many important therapeutic targets exert their function by complexationwith other proteins. The protein/protein interaction surfaces in many ofthese systems contain an inner core of hydrophobic side chainssurrounded by a wide ring of polar residues. The hydrophobic residuescontribute nearly all of the energetically favorable contacts, and hencethis cluster has been designated as a “hotspot” for engagement inprotein-protein interactions. Importantly, in the aforementionedcomplexes of naturally occurring small molecules with small presentingproteins, the small molecule provides a cluster of hydrophobicfunctionality akin to a hotspot, and the protein provides the ring ofmostly polar residues. In other words, presented small molecule systemsmimic the surface architecture employed widely in naturalprotein/protein interaction systems.

Compounds (e.g., macrocyclic compounds) of the invention are capable ofmodulating biological processes, for example through binding to apresenter protein (e.g., a member of the FKBP family, a member of thecyclophilin family, or PIN1) to form a presenter protein/compoundcomplex as described above which binds to the target protein to form atripartite complex. The presenter protein/compound complexes are able tomodulate biological processes through cooperative binding between thecompound and the presenter protein. Both the compound and presenterprotein have low affinity for the target protein alone, but thepresenter protein/compound complex has high affinity for the targetprotein. Cooperative binding can be determined by measurement of theburied surface area of the target protein that includes atoms from thecompound and/or presenter protein and/or by measurement of the freebinding energy contribution of the compound and/or presenter protein.Binding is considered cooperative if at least one atom from each of thecompound and presenter protein participates in binding with the targetprotein.

The binding of a presenter protein/compound complex and the targetprotein is achieved through formation of a combined binding siteincluding residues from both the presenter protein and compound thatallow for increased affinity that would not be possible with either thepresenter protein or compound alone. For example at least 20% (e.g., atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50%) of the total buried surface area of the target protein in thetripartite complex includes one or more atoms that participate inbinding to the compound and/or at least 20% (e.g., at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%) ofthe total buried surface area of the target protein in the tripartitecomplex includes one or more atoms that participate in binding to thepresenter protein. Alternatively, the compound contributes at least 10%(e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, or at least 90%) of the total binding free energy of the tripartitecomplex and/or the presenter protein contributes at least 10% (e.g., atleast 20% at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 60%, at least 70%, at least 80%, or atleast 90%) of the total binding free energy of the tripartite complex.

In some embodiments, a presenter protein/compound complex binds at aflat surface site on the target protein. In some embodiments, a compound(e.g., macrocyclic compound) in a presenter protein/compound complexbinds at a hydrophobic surface site on the target protein, e.g., a sitethat includes at least 50% hydrophobic residues. In some embodiments, atleast 70% of the binding interactions between one or more of the atomsof a compound and one or more atoms of the target protein are van derWaals and/or π-effect interactions. In certain embodiments, a presenterprotein/compound complex binds to the target protein at a site of anaturally occurring protein-protein interaction between the targetprotein and a protein that specifically binds the target protein. Insome embodiments, a presenter protein/compound complex does not bind atan active site of the target protein. In some embodiments, a presenterprotein/compound complex binds at an active site of the target protein.

A characteristic of compounds of the invention that form tripartitecomplexes with a presenter protein and the target protein is a lack ofmajor structural reorganization in the presenter protein/compoundcomplex compared to the tripartite complex. This lack of majorstructural reorganization results in a low entropic cost to reorganizeinto a configuration favorable for the formation of the tripartitecomplex once the presenter protein/compound complex has been formed. Forexample, threshold quantification of RMSD can be measured using thealign command in PyMOL version 1.7rc1 (Schrödinger LLC). Alternatively,RMSD can be calculated using the ExecutiveRMS parameter from thealgorithm LigAlign (J. Mol. Graphics and Modelling 2010, 29, 93-101). Insome embodiments, the structural organization of the compound (i.e., theaverage three dimensional configuration of the atoms and bonds of themolecule) is substantially unchanged in the tripartite complex comparedto the compound when in the presenter protein/compound complex beforebinding to the target protein (e.g., the root mean squared deviation(RMSD) of the two aligned structures is less than 1).

Utility and Administration

Compounds and presenter protein/compound complexes described herein areuseful in the methods of the invention and, while not bound by theory,are believed to exert their desirable effects through their ability tomodulate (e.g., positively or negatively modulate) the activity of thetarget protein through interaction with presenter proteins and thetarget protein.

CEP250 is a core centrisomal protein required for centriole-centriolecohesion during interphase of the cell cycle. Thus, the compounds of theinvention can be useful in the binding stabilization, or modulation ofthe activity of one or more components of the centrosome. The compoundsof the invention can also be used to modulate signal transductionpathways associated with CEP250, including, but not limited to,Hedgehog, Wnt, PDGFRalpha, and integrin signaling. The compounds of theinvention can also be useful in the treatment of diseases or disordersrelated to centrosome aberrations (e.g., cancer or ciliopathies) orHedgehog, Wnt, PDGFRalpha, or integrin signaling.

Centrioles are essential for the formation of cilia (e.g., motile ciliaor non-motile cilia). Thus, the compounds of the invention can be usedto modulate the activity of the primary cilium, for example, viainteraction with one or more components of the centrome. The compoundsof the invention can also be useful in the treatment of diseases ordisorders related to aberrant cilia function (i.e., ciliopathies).

The compounds described herein may be useful for the treatment ofcertain conditions such as ciliopathies, cancer, inflammation, andinfections (e.g., bacterial, fungal, or protozoal).

The compounds of the invention may be useful in the treatment ofciliopathies including, but not limited to, Alstrom syndrome,Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome,nephronophthisis, orofaciodigital syndrome 1, Senior-Loken syndrome,polycyctic kidney disease, primary ciliary dyskinesia (Kartagenersyndrome), asphyxiating thoracic dyslasia (Juene), Marden-Walkersyndrome, situs inversus/Isomerism, early embryonic death,hydrocephalus, polycystic liver disease, and retinal degeneration,agenesis of the corpus callowum, anencephaly, breathing abnormalities,cerebellar vermis hypoplasia, Dandy-Walker malformation, diabetes,Ellis-van Creveld syndrome, exencephaly, eye movement abnormalities,liver disease, hypoplasia of the corpus callosum, hypotonia, sterility,juvenile myoclonic epilepsy, obesity, polydactyly, posteriorencephalocele, respiratory dysfunction, renal cystic disease, retinitispigmentosa, sensorineural deafness, and spina bifida.

The compounds of the invention may be useful in the treatment cancers,including, but not limited to, basal cell carcinoma, squamous-cellcarcinoma, biliary tract cancer, bladder cancer, bone cancer, brain andother CNS cancer, cervical cancer, choriocarcinoma, connective tissuecancer, cancer of the digestive system, endometrial cancer, esophagealcancer, eye cancer, cancer of the head and neck, gastric cancer,intra-epithelial cancer, kidney cancer, larynx cancer, hairy cellleukemia, liver cancer, Hodgkin's and non-Hodgkin's lymphomas,medulloblastoma, melanoma, myeloma, neuroblastoma, oral cavity cancer(e.g. lip, tongue, mouth, pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer, renal cancer, cancer ofthe respiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem. The compounds of the invention may also be useful in thetreatment of inflammation related to rheumatoid arthritis, Sjogren'ssyndrome, coronary artery disease, peripheral vascular disease,hypertension, Alzheimer's disease and its variants, lupus erythematosus,chronic bronchitis, chronic sinusitis, benign prostatichypertrophy,prostate cancer, colon adenomas, colon cancer, cancer of the lung,lymphoma, and leukemia.

The compounds of the invention may also be useful in the treatment ofinfectious diseases such as candidasis or aspergillosis.

Kits

In some embodiments, the present invention relates to a kit forconveniently and effectively carrying out the methods in accordance withthe present invention. In general, the pharmaceutical pack or kitcomprises one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. Suchkits are especially suited for the delivery of solid oral forms such astablets or capsules. Such a kit preferably includes a number of unitdosages, and may also include a card having the dosages oriented in theorder of their intended use. If desired, for instance if the subjectsuffers from Alzheimer's disease, a memory aid can be provided, forexample in the form of numbers, letters, or other markings or with acalendar insert, designating the days in the treatment schedule in whichthe dosages can be administered. Alternatively, placebo dosages, orcalcium dietary supplements, either in a form similar to or distinctfrom the dosages of the pharmaceutical compositions, can be included toprovide a kit in which a dosage is taken every day. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Pharmaceutical Compositions

For use as treatment of human and animal subjects, the compounds of theinvention can be formulated as pharmaceutical or veterinarycompositions. Depending on the subject to be treated, the mode ofadministration, and the type of treatment desired—e.g., prevention,prophylaxis, or therapy—the compounds are formulated in ways consonantwith these parameters. A summary of such techniques is found inRemington: The Science and Practice of Pharmacy, 21^(st) Edition,Lippincott Williams & Wilkins, (2005); and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, each of which is incorporated hereinby reference.

Compounds described herein may be present in amounts totaling 1-95% byweight of the total weight of the composition. The composition may beprovided in a dosage form that is suitable for intraarticular, oral,parenteral (e.g., intravenous, intramuscular), rectal, cutaneous,subcutaneous, topical, transdermal, sublingual, nasal, vaginal,intravesicular, intraurethral, intrathecal, epidural, aural, or ocularadministration, or by injection, inhalation, or direct contact with thenasal, genitourinary, reproductive or oral mucosa. Thus, thepharmaceutical composition may be in the form of, e.g., tablets,capsules, pills, powders, granulates, suspensions, emulsions, solutions,gels including hydrogels, pastes, ointments, creams, plasters, drenches,osmotic delivery devices, suppositories, enemas, injectables, implants,sprays, preparations suitable for iontophoretic delivery, or aerosols.The compositions may be formulated according to conventionalpharmaceutical practice.

In general, for use in treatment, compounds described herein may be usedalone, or in combination with one or more other active agents. Anexample of other pharmaceuticals to combine with the compounds describedherein would include pharmaceuticals for the treatment of the sameindication. Another example of a potential pharmaceutical to combinewith compounds described herein would include pharmaceuticals for thetreatment of different yet associated or related symptoms orindications. Depending on the mode of administration, compounds will beformulated into suitable compositions to permit facile delivery. Eachcompound of a combination therapy may be formulated in a variety of waysthat are known in the art. For example, the first and second agents ofthe combination therapy may be formulated together or separately.Desirably, the first and second agents are formulated together for thesimultaneous or near simultaneous administration of the agents.

Compounds of the invention may be prepared and used as pharmaceuticalcompositions comprising an effective amount of a compound describedherein and a pharmaceutically acceptable carrier or excipient, as iswell known in the art. In some embodiments, a composition includes atleast two different pharmaceutically acceptable excipients or carriers.

Formulations may be prepared in a manner suitable for systemicadministration or topical or local administration. Systemic formulationsinclude those designed for injection (e.g., intramuscular, intravenousor subcutaneous injection) or may be prepared for transdermal,transmucosal, or oral administration. A formulation will generallyinclude diluents as well as, in some cases, adjuvants, buffers,preservatives and the like. Compounds can be administered also inliposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms asliquid solutions or suspensions or as solid forms suitable for solutionor suspension in liquid prior to injection or as emulsions. Suitableexcipients include, for example, water, saline, dextrose, glycerol andthe like. Such compositions may also contain amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like, such as, for example, sodium acetate, sorbitanmonolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See,for example, U.S. Pat. No. 5,624,677, which is herein incorporated byreference.

Systemic administration may also include relatively noninvasive methodssuch as the use of suppositories, transdermal patches, transmucosaldelivery and intranasal administration. Oral administration is alsosuitable for compounds of the invention. Suitable forms include syrups,capsules, and tablets, as is understood in the art.

Each compound of a combination therapy, as described herein, may beformulated in a variety of ways that are known in the art. For example,the first and second agents of the combination therapy may be formulatedtogether or separately.

The individually or separately formulated agents can be packagedtogether as a kit. Non-limiting examples include, but are not limitedto, kits that contain, e.g., two pills, a pill and a powder, asuppository and a liquid in a vial, two topical creams, etc. The kit caninclude optional components that aid in the administration of the unitdose to subjects, such as vials for reconstituting powder forms,syringes for injection, customized IV delivery systems, inhalers, etc.Additionally, the unit dose kit can contain instructions for preparationand administration of the compositions. The kit may be manufactured as asingle use unit dose for one subject, multiple uses for a particularsubject (at a constant dose or in which the individual compounds mayvary in potency as therapy progresses); or the kit may contain multipledoses suitable for administration to multiple subjects (“bulkpackaging”). The kit components may be assembled in cartons, blisterpacks, bottles, tubes, and the like.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystallinecellulose, starches including potato starch, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate); granulating and disintegrating agents (e.g., cellulosederivatives including microcrystalline cellulose, starches includingpotato starch, croscarmellose sodium, alginates, or alginic acid);binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

Two or more compounds may be mixed together in a tablet, capsule, orother vehicle, or may be partitioned. In one example, the first compoundis contained on the inside of the tablet, and the second compound is onthe outside, such that a substantial portion of the second compound isreleased prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluents (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders, granulates, and pellets may be prepared using the ingredientsmentioned above under tablets and capsules in a conventional mannerusing, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of thecompounds of the combination of the invention will depend on the natureof the compound, and can readily be determined by one skilled in theart. Typically, such dosage is normally about 0.001 mg to 2000 mg perday, desirably about 1 mg to 1000 mg per day, and more desirably about 5mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

Administration of each drug in a combination therapy, as describedherein, can, independently, be one to four times daily for one day toone year, and may even be for the life of the subject. Chronic,long-term administration may be indicated.

The following Examples are intended to illustrate the synthesis of arepresentative number of compounds and the use of these compounds forthe induction of chemotaxis and antifungal activity. Accordingly, theExamples are intended to illustrate but not to limit the invention.Additional compounds not specifically exemplified may be synthesizedusing conventional methods in combination with the methods describedherein.

EXAMPLES Example 1. General Fermentation and Isolation Protocols

Compounds synthesized by bacterial strains may be fermented and isolatedusing the following general protocol:

General Fermentation Protocol

Strains: Bacterial strains such as Streptomyces malaysiensis DSM41697,other producing species, or genetically modified derivatives producingFKBP ligands (Example: F1, F2, F3 or structurally similar compounds andtheir analogs) were propagated aseptically on a solid medium (Example:ISP4).

Working cell bank: Spores or mycelia derived from the cultures grown ona solid medium plate at 30° C. for 3-14 d were used to inoculate aliquid culture (Example: 40 ml ATCC172 liquid medium in an 250 mlErlenmeyer flask). The culture was incubated with shaking at 30° C. for2-3 d. The resulting cell suspension was mixed with sterile 50% glycerolgiving a mixture containing a final concentration of 15-25% glycerol.Aliquots (about 1 ml) of glycerol-mycelia mixture were stored at −80° C.in sterile cryovials until further use.

Primary seed culture: Primary seed cultures (Example: 40 mL ATCC172medium in a 250 mL Erlenmeyer flask) were inoculated with 1 mL workingcell bank suspension. Cultures were incubated on a shaker with a 2-inchthrow at 200-220 rpm for 2-3 d at 30° C.

Secondary seed culture: Secondary seed cultures (Example: 100-200 mLATCC172 in an 500 mL Erlenmeyer flask) were inoculated with the primaryseed cultures (5% v/v) and incubated as described above with variousincubation periods of time (Example: 18-48 h).

Production fermentation in flasks: Production fermentation was done in a1.8 L Fernbach or Erlenmeyer flask containing 0.5 L production mediumsupporting biosynthesis of these compounds (Example: Medium 8430 or itsderivatives). The culture was inoculated with a seed culture prepared asdescribed above at 2-5% (v/v), and incubated as described aboveconditions for 3-7 d.

Production fermentation in bioreactors: Production fermentation was donein a bioreactor (7.5 L capacity, New Brunswick Scientific, NJ, USA)controlled by a BioFlo 300 module. The bioreactor containing 5 L ofsterilized medium (Example: 8430 and its derivatives) was inoculatedwith a seed culture (2-5%, v/v) and incubated for 3-7 d with or withoutcontrolled parameters such as dissolved oxygen amounts (Example:10-50%), propeller speed (Example: 200-500 rpm), pH (Example: pH4.5-7.0), temperature (Example: 25-35° C.), and nutrient feeding whenappropriate.

ISP4 (per liter) Soluble Starch 10.0 g Dipotassium Phosphate 1.0 gMagnesium Sulfate USP 1.0 g Sodium Chloride 1.0 g Ammonium Sulfate 2.0 gCalcium Carbonate 2.0 g Ferrous Sulfate 1.0 mg Manganous Chloride 1.0 mgZinc Sulfate 1.0 mg Agar 20.0 g

TABLE 3 ATCC #172 media (per liter) Yeast extract  5 g Difco SolubleStarch 20 g Dextrose 10 g NZ Amine A  5 g Calcium Carbonate  3 g Adddistilled water to 1000 mL, no pH adjustment.

TABLE 4 8430 Medium Component Amount Pharmamedia or Proflo powder (ADM)10 g D-Mannitol 20 g Yeast extract 1.0 g KH₂PO₄ 0.10 g MES buffer,hemi-Na+ salt (100 mM final) 20.67 g (adjust media to pH 6.5 final with5N NaOH) MgSO₄—7H₂O (Anh.) 0.05 g/L CaCl₂—2H₂O 0.02 g/L R2 traceelements solution* 2 mL Add distilled water to 1000 mL. Proflo oilcontaining dominantly oleate and palmitate was added (4 mL/L) as anantifoam agent.

TABLE 5 * R2 trace element solution. Amount Element (mg/L) ZnSO₄—7H₂O 40FeCl₃—6H₂O 200 CuCl₂—2H₂O 10 MnCl2—2 H₂O 10 Na₂B₄O₇—10H₂O 10(NH₄)₆Mo₇O₂₄—4H₂O 10

General Isolation Protocol

Fermentation broth of a strain producing specific compounds wasseparated to supernatant and microbial pellets by centrifugation. Targetcompounds in the supernatant can be extracted either with partitionextraction using water-immiscible solvents such as dichloromethane(DCM), ethyl acetate (EtOAc), etc or with solid phase extraction bymixing with non-polar resins such as HP20, HP20ss, etc. The targetcompounds in the pellets can be extracted repeatedly (4×) using ethylEtOAc-methanol (9:1, v/v). The microbial extracts are pooled inpreparation for concentrating in vacuo. To this extract can be added thematerial eluted from the HP20 beads (using organic solvents such asmethanol (MeOH), DCM, acetonitrile, isopropanol (IPA), etc) and/or theorganic phase of the liquid/liquid extraction of the originalsupernatant.

The combined extracts are filtered through Celite and dried in vacuoyielding a primary crude and this material is weighed. The primary crudeis dissolved in minimal 100% MeOH or a mixture of DCM andtetrahydrofuran (THF). To this a binding medium such as silica gelpowder is added to the flask and re-dried in vacuo for normal-phasesilica gel column chromatography. The ratio of crude to silica gel inthe column bed is preferably ca. 1:5 (wt/wt). The crude material can befractionated over a RediSep® Normal-phase Silica Flash Column using stepgradients, linear gradients or isocratic elution conditions. Elutionsolvents can include hexane, heptane, ethyl acetate, ethanol, acetone,isopropanol, or other organic solvents, or combination. Fractions withenriched target compound(s) are pooled and dried for furtherpurification after LC/MS analysis and/or Thin Layer Chromatography (TLC)analysis.

Further purification could be achieved via normal-phase or specificprep-HPLC columns such as Waters Spherisorb CN, Waters Prep Silica, orKromacil 60-5DIOL. Elution solvents can also include hexane, heptane,ethyl acetate, ethanol, acetone, isopropanol, or other organic solvents,or combination. Fractions with enriched or pure target compound(s) arepooled and dried for further workup after LC/MS analysis and/or ThinLayer Chromatography (TLC) analysis.

Additional purification could be achieved various reverse-phaseprep-HPLC depending on the complexity of the enriched material andtarget compounds' properties such as polarity, solubility, etc.Reverse-phase Prep-H PLC columns employed for separation include WatersSunfire Prep C18 OBD, Waters Xbridge Prep C18 OBD, Kromacil C4, ThermoAcclaim Polar Advantage 2, and Phenomenex Luna C18. Common solventsystems are a mixture of water and acetonitrile or methanol without orwith 0.1% formic acid or 0.01% trifluoroacetic acid modifiers or 25 mMammonium formate buffer. The elution mode can be either linear gradientor isocratic. Fractions with pure target compound(s) are pooled anddried for further workup after LC/MS analysis and/or Thin LayerChromatography (TLC) analysis.

Fractions containing pure compounds are subjected to workup and dryingprocess to obtain pure solid material. Certain target compounds can beextracted with ethyl acetate or dichloromethane from aqueous matrixafter reverse-phase column chromatographic purification. Solvent removaland drying techniques include rotavap, speedvac, and lyophilization.Purity and chemical structure of purified target compounds aredetermined by LC-MS(/MS) and NMR techniques.

Example 2. Isolation of Compound 2 and Compound 3

10 L fermentation broth of a Streptomyces malaysiensis strain (NRRLB-24313; ATCC BAA-13; DSM 41697; JCM 10672; KCTC 9934; NBRC 16446; CGMCC4.1900; IFO 16448) producing Compound 1 (target mass 595), Compound 2(target mass 609), and Compound 3 (target mass 623) was separated bycentrifugation. Compound 1 and Compound 2 are present in both theclarified broth and microbial pellets. Target compounds in thesupernatant were extracted once with EtOAc at a ratio of volume (1:1,v/v). The pellets were extracted 3 times with 1.5 L of EtOAc-MeOH (9:1,v/v) stirring with an overhead stirrer for 1 h-1.5 h for eachextraction. The organic extracts were filtered through Celite. Thecombined filtrates were evaporated at 35° C. until dryness to afford ca.30 g of crude extract. The residue was then dissolved in 90 mL ofDCM-THF (80:20, v/v), and to this 60 g of silica gel were added anddried in vacuo at 35° C. The dried residue/silica mixture was loadedonto a 120 g RediSep silica gold cartridge. Compounds were eluted with100% heptane to heptane-EtOAc (6:4, v/v) with a linear gradient over 30min at 85 mL/min and collected with 50 mL per fraction on a TeledyneISCO Combiflash Rf instrument.

By TLC, Compound 2 enriched fractions were eluted at 20% to 30% EtOAc inheptane. The pooled fraction was then concentrated at 35° C. to provide900 mg of enriched F2 material which was further re-purified on a silicagel cartridge. Ca. 1 mL of DCM was used to dissolve the fraction and 1.8g of silica gel was added. The dried mixture was loaded onto a 80 gRediSep silica gold cartridge. Compounds were eluted with 100% heptaneto heptane-EtOAc (6:4, v/v) with a linear gradient over 30 min at 60mL/min and collected with 50 mL per fraction. By TLC, pure fractions25-28 were combined for solvent removal in vacuo at 35° C. to obtain 300mg of pure Compound 2 (beta-form) for structure elucidation andbiological tests.

Compound 2: ¹H NMR (500 MHz, Benzene-d₆) δ 7.20-7.13 (m, 4H), 7.0-7.05(m, 1H), 5.82 (s, 1H), 5.79-5.69 (m, 2H), 5.51 (m, 1H), 5.46-5.35 (m,3H), 4.60 (d, J=12 Hz, 1H), 3.98-3.90 (m, 1H), 3.63 (dqd, J=13, 6.5, 3.0Hz, 1H), 3.22 (d, J=3.6 Hz, 1H), 3.07 (td, J=12, 2.8 Hz, 1H), 3.00 (t,J=9.9 Hz, 1H), 2.93 (dd, J=13, 4.4 Hz, 1H), 2.63-2.54 (m, 3H), 2.20 (d,J=13 Hz, 1H), 2.11-2.03 (m, 1H), 1.99-1.86 (m, 2H), 1.79-1.71 (m, 1H),1.68-1.60 (m, 1H), 1.51-1.47 (m, 1H), 1.45 (d, J=6.6 Hz, 3H), 1.37 (m,4H), 1.31 (m, 1H), 1.30 (d, J=6.6 Hz, 3H), 1.29-1.22 (m, 2H), 1.16-1.08(m, 1H), 1.04-0.94 (m, 1H), 0.82 (t, J=7.4 Hz, 3H), 0.69 (d, J=6.7 Hz,3H). ¹³C NMR (125 MHz, Benzene-d₆) δ 209.9, 169.7, 167.5, 141.3, 132.2,129.6, 129.4, 128.7, 128.0, 127.7, 126.4, 98.2, 79.7, 75.5, 71.1, 51.9,46.9, 44.2, 44.0, 40.4, 36.2, 35.3, 35.3, 35.2, 34.0, 33.3, 25.4, 25.3,22.5, 21.1, 17.4, 17.1, 11.6, 9.7. HR-MS [M+Na]⁺: calc [C₃₆H₅₁NO₇+Na]⁺632.3563, obs 632.3569 (A=0.9 ppm).

By TLC and LC-MS analysis, Compound 3 enriched fractions were eluted at30% to 40% EtOAc in heptane. The pooled fraction was then concentratedat 35° C. to provide 500 mg of enriched Compound 3 material which wasfurther re-purified by reverse-phase prep-HPLC on a Thermo PolarAdvantage II column (5 μm, 250×21.2 mm). Prep-HPLC conditions included70% acetonitrile in water plus 0.1% formic acid, isocratic elution modeat 15 mL/min, 254 nm. The enriched Compound 3 sample was dissolved in 10mL methanol for repeatable 10 injections. Target Compound 3 peak at 23.5minute was collected. After extraction with EtOAc from prep-HPLC pooledfractions and organic solvent removal in vacuo, 250 mg of pure Compound3 were obtained. Its chemical structure was subsequently determined byvarious LC-MS and NMR techniques.

Compound 3: ¹H NMR (500 MHz, Benzene-cis, 1:1 mixture of rotamers) δ7.30 (m, 1H), 7.20-7.10 (m, 6H), 7.10-7.06 (m, 3H), 7.00 (m, 2H),5.65-5.55 (m, 2H), 5.45 (m, 1H), 5.25-5.15 (m, 2H), 4.98 (dd, J=15, 7.3Hz, 1H), 4.89 (dd, J=8.9, 5.0 Hz, 1H), 4.67 (dd, J=15, 8.8 Hz, 1H), 4.45(m, 2H), 4.20 (m, 1H), 4.13 (m, 1H), 3.87 (m, 1H), 3.57 (m, 2H),3.35-3.05 (m, 3H), 2.72 (m, 2H), 2.65-2.50 (m, 2H), 2.50-2.30 (m, 6H),2.08 (m, 1H), 1.93 (m, 1H), 1.80-0.90 (m, 50H) [1.71 (d, J=6.8 Hz, 3H),1.54 (d, J=6.8 Hz, 3H)], 1.24 (d, J=6.5 Hz, 3H), 1.18 (d, J=6.6 Hz, 3H),1.08 (d, J=6.8 Hz, 3H), 1.01 (m, J=6.7 Hz, 3H)], 0.73 (t, J=7.5 Hz, 3H),0.69 (t, J=7.5 Hz, 3H). ¹³C NMR (125 MHz, Benzene-d₆) δ 201.5, 199.9,197.8, 191.6, 170.4, 169.5, 166.8, 166.6, 145.4, 144.8, 140.6, 140.5,133.9, 131.3, 129.7, 129.4, 129.3, 128.8, 128.8, 128.4, 128.3, 126.6,126.5, 126.0, 100.0, 99.3, 80.6, 78.2, 73.1, 72.4, 71.7, 70.6, 57.1,52.6, 51.9, 51.1, 45.8, 45.4, 44.2, 42.5, 42.2, 39.8, 35.8, 35.7, 35.6,34.1, 33.6, 33.4, 29.9, 29.9, 29.3, 28.2, 27.4, 27.1, 25.1, 25.1, 22.3,22.2, 21.3, 21.2, 16.6, 16.2, 14.6, 13.7, 11.2, 11.1, 10.6, 9.5. HR-MS[M+H]⁺: calc [C₃₆H₄₉NO₈+H]⁺ 624.3536, obs 624.3547 (A=1.8 ppm).

Example 3. Isolation of Compound 9

10 L of fermentation broth produced from a Streptomyces malaysiensisstrain were centrifuged to obtain the pellets and supernatant. Thepellets were extracted 3 times with 1.5 L of EtOAc-MeOH (9:1, v/v). Theorganic solvents were combined and concentrated in vacuo to obtain 1.8 gof crude extract. To this, 2 mL of Heptane-THF (4:1, v/v) was added todissolve and 2 g of Celite were then added to obtain the dried mixtureafter removal of solvents on a rotavapor at 30° C. The driedresidue/celite mixture was loaded onto a 40 g RediSep silica goldcartridge for column chromatography. Compounds were fractionated with alinear gradient elution from 100% n-heptane to 40% EtOAc in heptane(v/v) over 25 min at 20 mL/min collected with 50 mL per fraction.Compound 9 (target mass 607) was primarily enriched in Fraction 14identified by LC-MS analysis. Fraction 14 was then dried in vacuo at 30°C. to afford 17.8 mg solid material which was further purified byprep-HPLC on a Thermo Polar Advantage II column (5 μm, 250×21.2 mm).Prep-HPLC conditions included 90% acetonitrile in water plus 0.1% formicacid, isocratic elution mode at 15 mL/min, 254 nm. The sample wasdissolved in 1.78 mL methanol for repeatable 5 injections. Target F22peak at 11.5 minute was collected. After solvent removal in vacuo, 3.64mg of pure F22 was obtained. Its chemical structure was subsequentlydetermined by various LC-MS/MS and NMR techniques.

Example 4. Synthesis of Selected Compounds Instrumentation:

Purification was performed on HPLC preparative using Agilent SD-1system.Electrospray LC/MS analysis was performed using an Agilent 1260 Infinitysystem equipped with an Agilent 1260 series LC pump. The methods usedwere:

Analytical HPLC Method 1:

Agilent Zorbax Extend C-18 reverse phase column (2.1×50 mm), 1.8 μm:

Solvent A: Water+0.1% Formic Acid Solvent B: Acetonitrile+0.1% FormicAcid

Flow rate: 0.5 mL/minInjection volume: 5 μLColumn temperature: 40° C.

Gradient:

Time, min % A % B 0 95 5 3 30 70 10 0 100 13 0 100 14 95 5 16 95 5

Analytical HPLC Method 2: ThermoScientific Acclaim, Polar Advantage II,4.6×150 mm, 5 μm Solvent A: Water+0.1% Formic Acid Solvent B:Acetonitrile+0.1% Formic Acid

Flow rate: 0.8 mL/minInjection volume: 5 μLColumn temperature: 40° C.

Isocratic:

Time, min % A % B 0 20 80 6 20 80 7 5 95 9 5 95 10 20 80 12 20 80Electrospray UHPLC/MS was performed using an Agilent 1290 Infinitysystem equipped with an Agilent1290 series LC pump. The columns used were the same.

Analytical UHPLC Method 1:

Agilent Zorbax Extend C-18 reverse phase column (2.1×50 mm), 1.8 μm:

Solvent A: Water+0.1% Formic Acid Solvent B: Acetonitrile+0.1% FormicAcid

Flow rate: 0.5 mL/minInjection volume: 5 μLColumn temperature: 40° C.

Gradient:

Time, min % A % B 0 95.24 4.76 5.21 30.19 69.81 9.66 9.04 90.96 10.5 0100 11.5 0 100 12 95.24 4.76 13 95.24 4.76

Purification Method A:

Performed using an ACCLAIM Polar Advantage II (21.2×250 mm) column. Flowrate 17 mL/min, isocratic 70% B. Solvent A was 0.1% aqueous formic acid,solvent B was 100% acetonitrile containing 0.1% formic acid.

Synthesis of Compound 6 Synthesis of (2S)-1-((4R,7S)-7-((2R,3S,4R,11S,12R)-12-benzyl-3,11-dihydroxy-4-methyltetradecan-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylicacid C-11 lactone

To a mixture of(2S)-1-((4R,7S)-7-((2R,3S,4R,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyltetradeca-6,9-dien-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylicacid C-11 lactone (5 mg, 8.2 umol) and 10% palladium on carbon (2 mg)and a stirrer bead under nitrogen was added ethyl acetate (1 mL). Theflask was charged with hydrogen and stirred vigorously for 1.5 hr. Theatmosphere of hydrogen was replaced with nitrogen and the reactionfiltered through celite. The celite pad was washed with more ethylacetate and the solvent evaporated in vacuo. The residue was purified bychromatography on silica gel, gradient elution ethyl acetate:hexanes40:60 to 100:0 to afford the title compound.

1H NMR (CDCl3, 500 MHz): δ 7.28 (m, 2H), 7.19 (m, 1H), 7.13 (d, J=6.98Hz, 2H), 5.65 (s, 1H), 5.26 (d, J=4.92 Hz, 1H), 5.11 (m, 1H), 4.67 (d,J=13.02 Hz, 1H), 4.02 (dd, J=10.67, 1.13 Hz, 1H), 3.35 (m, 1H),3.23-3.10 (m, 2H), 2.72 (dd, J=13.85, 5.50 Hz, 1H), 2.50 (dd, J=13.93,9.25 Hz, 1H), 2.39 (m, 1H), 1.95-1.73 (m, 5H), 1.71-1.15 (m, 25H), 1.03(d, J=6.71 Hz, 3H), 0.85 (t, J=7.42 Hz, 3H), 0.79 (d, J=6.82 Hz, 3H)ppm.

13C NMR (CDCl3, 500 MHz): δ 210.5, 170.3, 167.4, 140.5, 129.0, 128.3,126.0, 97.8, 79.1, 76.9, 71.1, 52.0, 45.9, 43.9, 43.5, 39.9, 36.3, 35.1,33.1, 32.3, 31.9, 29.1, 27.9, 27.1, 25.8, 25.1, 23.4, 22.1, 21.1, 20.0,17.0, 16.6, 11.5, 8.9 ppm.

MS (ESI): calculated for (C36H55NO7+H)+614.4057, found 614.4066.

Synthesis of Compound 10 Synthesis of(S)-1-(2-((2R,3R,6S)-6-((2R,3R,4S,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradeca-6,9-dien-2-yl)-2-hydroxy-3-methyltetrahydro-2H-pyran-2-yl)-2-oxoacetyl)piperidine-2-carboxylicacid C-11 lactone

To a solution of Compound 3 (24.2 mg, 36.7 μmol) in ethyl acetate (1 mL)under nitrogen was added 10% Pd/C (12 mg, 50% w/w). The flask wascharged with hydrogen and the suspension was stirred at room temperaturefor 30 min. The hydrogen was replaced with nitrogen and the reactionmixture was then filtered through celite. The filtrate was concentratedunder vacuum to give 24 mg of crude product, of which, a portion waspurified using Method A to afford the tetrahydro Compound 3 as a whitesolid (11 mg, 47.8%). TLC: (50/50 heptane/ethyl acetate) Rf=0.45.

¹H NMR (400 MHz, C₆D₆, 1:0.3 mixture of rotamers, asterisk (*) denotespeaks associated with the minor isomer) δ 7.25-7.0 (m, 5H), 6.18* (s,1H), 5.33-5.28 (m, 2H), 5.11* (d, J=12 Hz, 1H), 4.92* (m, 1H), 4.45* (d,J=12 Hz, 1H), 4.24 (m, 1H), 4.06* (td, J=8 Hz, 1H), 3.40 (dd, J=4 Hz,1H), 3.88 (t, J=8 Hz, 1H), 3.65 (d, J=12 Hz, 1H), 3.30 (td, J=12 Hz,1H), 3.02* (td, J=12, 4 Hz, 1H), 2.84* (m, 1H), 2.74 (dd, 16, 8 Hz, 1H),2.65 (q, 8 Hz, 1H), 2.61-2.48 (m, 2H), 2.38-2.09 (m, 5H), 1.73-1.54 (m,6H), 1.47-1.02 (m, 28H), 0.90 (m, 4H), 0.80 (t, J=8 Hz, 3H), 0.73* (t,J=8 Hz, 3H) ppm.

¹³C NMR (400 MHz, C₆D₆) δ: 212.59, 197.51, 170.64, 166.70, 140.93,129.39, 128.77, 128.17, 127.94, 126.43, 99.30, 76.54, 72.64, 71.61,52.46, 51.24, 46.46, 45.30, 41.62, 40.82, 36.20, 35.31, 32.09, 29.96,29.47, 27.67, 26.17, 25.71, 24.92, 22.57, 21.78, 21.59, 16.59, 13.68,11.49, 10.37 ppm. MS (ESI): calculated for (C₃₆H₅₃NO₈+Na)⁺ 650.37, found650.3.

Synthesis of Compound 11 Synthesis of (2S)-1-((4R,7S)-7-((2R,3R,4S,11S,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradecan-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylicacid C-11 lactone

tert-Butyldimethylsilyl trifluoromethanesulfonate (6.9 μL, 30.1 μmol)was added by syringe to an ice-cooled solution of(S)-1-(2-((2R,3R,6S)-6-((2R,3R,4S,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradeca-6,9-dien-2-yl)-2-hydroxy-3-methyltetrahydro-2H-pyran-2-yl)-2-oxoacetyl)piperidine-2-carboxylicacid C-11 lactone-Compound 10 (18.8 mg, 30.1 μL) and triethylamine (4.0μL, 30.1 μL) in dichloromethane (2 mL) under nitrogen. The resultingsolution was stirred at 0° C. for 15 min and was then allowed to warm toroom temperature for 2 h. The reaction was cooled to 0° C. and a secondportion of triethylamine (4.0 μL, 30.1 μL) and tert-butyldimethylsilyltrifluoromethanesulfonate (6.9 μL, 30.1 μmol) was added. The reactionwas again allowed to warm to room temperature and stirred under nitrogen16 h. Dichloromethane (10 mL) and 0.5 M aqueous sodium bicarbonatesolution (10 mL) were added and the organic layer was separated andwashed with 5% brine solution (10 mL), dried over anhydrous sodiumsulfate, filtered, and the filtrate concentrated in vacuo. The crudeproduct was purified using method A to afford the starting material as awhite solid (2.52 mg) and the title compound (4.51 mg) as a white solid.

¹H NMR (400 MHz, C₆D₆) δ 7.26-7.16 (m, 4H), 7.07 (tt, J=6.4, 2 Hz, 1H),5.58 (s, 1H), 5.39 (d, J=4.8 Hz, 1H), 5.17 (m, 1H), 4.67 (d, J=12.4 Hz,1H), 4.29 (d, J=10.8 Hz, 1H), 3.42 (m, 1H), 3.06 (td, J=11.2, 2.8 Hz,1H), 2.92 (t, J=10 Hz, 1H), 2.85 (m, 1H), 2.75 (s, 1H), 2.66 (dd, J=14,5.6 Hz, 1H), 2.52 (dd, J=14, 9.2 Hz, 1H), 2.35 (m, 1H), 2.28 (m, 1H),1.84 (m, 2H), 1.68-1.59 (m, 2H), 1.46-1.06 (m, 23H), 0.88 (m, 4H), 0.82(t, 3H, J=7.2 Hz) ppm.

¹³C NMR (400 MHz, C₆D₆) δ: 226.15, 210.53, 209.8, 179.03, 167.59,140.84, 129.40, 128.77, 128.18, 127.9, 126.45, 98.16, 79.21, 76.85,70.48, 52.20, 46.07, 44.46, 43.88, 42.76, 36.67, 35.30, 35.14, 32.88,30.53, 27.94, 25.49, 25.32, 24.57, 22.39, 21.36, 20.72, 16.98, 15.16,11.68, 9.08 ppm.

MS (ESI): calculated for (C₃₆H₅₃NO₈+Na)⁺ 650.37, found 650.3.

Example 5. Alternative Synthesis of Compounds of Formula I

Compounds of Formula I may be synthesis as shown in Scheme 1 below.

Treatment of Intermediates B, C, or D with 1-phenyl-butyl Grignardreagent results in the elongated alcohol, which may be protected withMOM-Cl. Treatment of the alcohol with butyl lithium in the presence ofIntermediate A results in the tetra-alcohol, which may be protected withTBS-Cl. DDQ deprotection and Dess-Martin oxidation provides the terminalaldehyde, which when reacted with a TMS-enol ether provides the ester.

Intermediate A may be synthesized as shown in Scheme 2 below:

Intermediates B, C, and D may be synthesized as shown in Scheme 3 below.

Macrocyclization of the compounds may be performed as shown in Scheme 4below.

MOM deprotection and acylation provides the N-protected pipecolic ester.Fmoc deprotection and HCTU amidation provides the macrocycle, which maybe deprotected and oxidized to provide the di- or triketone.

The final cyclization of the compounds may be performed as shown inScheme 5 below.

Deprotection of the free alcohols with TBAF provides the six-memberedand/or seven-membered ring containing compounds of Formula I.

Example 6. Binding of Compounds to FKBP12

The binding of compounds of the invention to FKBP12 can be determinedusing the following protocol.

General Protocol

This protocol utilizes Perkin Elmers AlphaLISA technology platform todetect FKBP binders by measuring the inhibition of binding ofbiotinylated FK506 to FLAG tagged FKBP12.

Reagents:

10×TBST Buffer (Boston BioProducts IBB-181), Biotinylated FK506(in-house), FLAG tagged FKBP (in-house); anti-FLAG Donor beads (PerkinElmer AS103) and Streptavidin Acceptor beads (Perkin Elmer AL125);Compounds in DMSO (in-house), FK506.

Equipment:

Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat PipettorSupplies: White 96-well Corning ½ area plates (Cat #3642), 96-wellpolypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tipsfor Janus MTD Head pipettor.

Experimental Protocol/Description of Assay: Add 20 uL of 12.5 nMFKBP-FLAG working stock to each well of the 96-well plate. Add 1 uL oftest compound (100% DMSO) to each well of the plate using the Janus MTDhead and P20 tips (except control wells). Add 1 uL of DMSO to negativecontrol wells and 1 uL of 50 uM FK506 solution to positive controlwells. In the dark, add 20 uL of combined Donor/Acceptor beads to eachwell. Incubate in the dark for 30 minutes at room temperature. In thedark, add 10 uL of 5 nM biotinylated FK506 working stock to each well.Incubate in the dark for 60 minutes at room temperature. Protect platefrom light until reading on Biotek Synergy2 Plate Reader; Alphalisa96-well protocol (680 excitation/615 emission).

Results:

The FKBP12 binding for selected compounds was determined as shown inTable 8.

TABLE 7 FKBP12 Binding Binding affinity to FKBP12 (by # displacement ofFK506) Compound 1 355 nM Compound 2 1.5 nM Compound 3 0.34 nM Compound 44.8 nM Compound 5 18.8 nM Compound 6 0.51 nM Compound 7 0.95 nM Compound8 1.1 nM Compound 9 785 nM Compound 10 0.21 nM

Example 7. SPR Protocol to Measure Binding of a Compound to FKBP12

This protocol utilizes Surface Plasmon Resonance (SPR) as a method todetermine kinetics (K_(D), K_(a), K_(d)) for the binding of compound(analyte) to immobilized FKBP12 (ligand).

Reagents:

Compound in 100% DMSO (in-house), 10×HBS-P+ buffer (GE HealthcareBR-1006-71), Assay buffer (1×HBS-P+ buffer, 1% DMSO), 12×HIS taggedFKBP12 (in-house).

Equipment:

BIACORE™ X100 (GE Healthcare)

Supplies:

NTA Sensor chip (GE Healthcare BR-1000-34)

Experimental Protocol:

Experiments are performed at 25° C. Stock solution of 12×HIS taggedFKBP12 is diluted to 100 nM in assay buffer (1% DMSO final).Approximately 500-600 RU of FKBP12 is immobilized on one of two flowcells of an activated NTA chip. The second flow cell is not activated asa reference for non-specific interaction of the analyte to the sensorchip. Various concentrations of compound (1 nM-1 μM range), seriallydiluted into the same assay buffer (1% DMSO final), are injected ontothe FKBP12 surface and reference surface at a flow rate of 10 μl/min.The surface is regenerated between analyte injections with 350 mM EDTA.

Data Fitting:

The BiaEvaluation software program is used for data fitting. All data isreference subtracted against both the reference flow cell and a bufferinjection. For kinetic analyses, data is locally fit to a 1:1interaction model.

TABLE 8 FKBP12 Binding Data # SPR affinity to FKBP12: K_(D) Compound 171.5 nM Compound 2 12.6 nM Compound 3 1.3 nM Compound 4 23.1 nM Compound5 7 nM Compound 6 21.5 nM

Example 8. Determination of Binding of Compound 2 and Compound 6 toFKBP12 by SPR

This protocol utilizes Surface Plasmon Resonance (SPR) as a method todetermine kinetics (K_(D), K_(a), K_(d)) for the binding of Compound 2and Compound 6 (analyte) to immobilized FKBP12 (ligand).

Reagents:

Compound 2 and Compound 6 in 100% DMSO (in-house), 10×HBS-P+ buffer (GEHealthcare BR-1006-71), Assay buffer (1×HBS-P+ buffer, 1% DMSO), 12×HIStagged FKBP12 (in-house).

Equipment:

BIACORE™ X100 (GE Healthcare)

Supplies:

NTA Sensor chip (GE Healthcare BR-1000-34)

Experimental Protocol:

Experiments are performed at 25° C. Stock solution of 12×HIS taggedFKBP12 is diluted to 100 nM in assay buffer (1% DMSO final).Approximately 500-600 RU of FKBP12 is immobilized on one of two flowcells of an activated NTA chip. The second flow cell is not activated asa reference for non-specific interaction of the analyte to the sensorchip. Various concentrations of Compound 2 or Compound 6 (1 nM-1 μMrange), serially diluted into the same assay buffer (1% DMSO final), areinjected onto the FKBP12 surface and reference surface at a flow rate of10 μl/min. The surface is regenerated between analyte injections with350 mM EDTA.

Data Fitting:

The BiaEvaluation software program is used for data fitting. All data isreference subtracted against both the reference flow cell and a bufferinjection. For kinetic analyses, data is locally fit to a 1:1interaction model.

Results:

The values for binding of Compound 2 to FKBP 12 are: K_(a) (1/Ms):4.50×10⁴; K_(d) (1/s): 5.94×10⁻⁴; and K_(D): 13.2 nM.

The values for binding of Compound 6 to FKBP12 are: K_(a) (1/Ms):5.67×10⁵; K_(d) (1/s): 8.8×10⁻³; and K_(D): 15.6 nM.

Example 9. Determination of Cell Permeability of Compounds

Cell permeability of compounds can be determined using the followingprotocol.

General Protocol

This protocol utilizes a modified FKBP or cyclophilin destabilizingmutant to determine the bioactivity of FKBP binding compounds orcyclophilin binding compounds in whole cell assay.

Reagents:

DMEM, DMEM without Phenol Red, 10% FBS, 1× Sodium Pyruvate, 1× Glutamax.Add 125 ul of media with compound per well.

Equipment:

Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat PipettorSupplies: White 96-well Corning ½ area plates (Cat #3642), 96-wellpolypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tipsfor Janus MTD Head pipettor.

Experimental Protocol/Description of Assay: Plate HeLa-FKBP12 cells (forFKBP binding compounds) or HeLa-CyclophilinA cells (for cyclophilinbinding compounds) and seed overnight at 5k/well (approximately −18hrs.). Using a multi-channel pipet, take out the old media and add ˜125ul of new media with compounds. Compounds are diluted using DMEM withoutPhenol Red, 10% FBS, 1× Sodium Pyruvate, lx Glutamax. Add 125 ul ofmedia with compound per well. Cells are treated with compounds atconcentration: 30, 10, 3.33, 1.11, 0.37, 0.12, 0.04 and 0.013 uM. Timepoints are taken at 72 hrs and plate read using plate reader withexcitation/emission: 575/620.

Calculation:

Cell binding/permeability is calculated in fold-change (Total RFU oftreated samples/total RFU of DMSO treated samples or total RFU abovebackground (Total RFU minus total RFU of DMSO treated samples).

Results:

Cell permeability data was gathered for selected compounds as shown inTable 10.

TABLE 9 Biosensor Permeability # Biosensor Permeability IC50 valueCompound 2 <1 uM Compound 4 <1 uM Compound 7 <1 uM Compound 8 <1 uMCompound 9 >1 uM

Example 10. Binding of a Presenter Protein/Compound Complex to a TargetProtein

The binding of a presenter protein/compound complex of the invention toa target protein can be determined using the following protocol.

General Protocol for FKBP 12 Complexes

This protocol utilizes Perkin Elmers AlphaLISA technology platform todetect compounds by measuring the binding of 6×HIS tagged targetprotein+FLAG tagged FKBP12 and FKBP binding compound.

Reagents:

10×TBST Buffer (Boston BioProducts IBB-181), MgCl₂ (Sigman), 6×HIStagged target protein (in-house), FLAG tagged FKBP12 (in-house);anti-FLAG Donor beads (PerkinElmer AS103) and Streptavidin Acceptorbeads (PerkinElmer AL125); Compounds in DMSO (in-house), FK506.

Equipment:

Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

Supplies: White 96-well Corning ½ area plates (Cat #3642), 96-wellpolypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tipsfor Janus MTD Head pipettor.

Experimental Protocol/Description of Assay:

Add 20 uL of 250 nM 6×HIS tagged target protein working stock to eachwell of the 96-well plate. Add 1 uL of test compound (100% DMSO) to eachwell of the plate using the Janus MTD head and P20 tips (except controlwells). Add 1 uL of DMSO to control wells. In the dark, add 20 uL ofcombined Donor/Acceptor beads to each well. Incubate in the dark for 30minutes at room temperature. In the dark, add 10 uL of 10 uM Flag taggedFKBP12 working stock to each well. Incubate in the dark for 60 minutesat room temperature. Protect plate from light until reading on BiotekSynergy2 Plate Reader; Alphalisa 96-well protocol (680 excitation/615emission).

Example 11. Determination of Binding of Presenter Protein/CompoundComplexes to Target Proteins by SPR

This protocol utilizes Surface Plasmon Resonance (SPR) as a method todetermine kinetics (K_(D), K_(a), K_(d)) for the binding of mammaliantarget protein (analyte) to immobilized FKBP12-compound binary complex(ligand).

Reagents:

Compound in 100% DMSO (in-house), 10×HBS-P+ buffer (GE HealthcareBR-1006-71), Assay buffer (1×HBS-P+ buffer, 1% DMSO, 1 μM F2), 12×HIStagged FKBP12 (in-house), mammalian target protein (in-house).

Equipment:

BIACORE™ X100 (GE Healthcare)

Supplies:

NTA Sensor chip (GE Healthcare BR-1000-34)

Experimental Protocol:

Experiments are performed at 25° C. Stock solution of 12×HIS taggedFKBP12 is diluted to 100 nM in assay buffer containing 1 μM compound (1%DMSO final). Approximately 200-400 RU of FKBP12 is immobilized on one oftwo flow cells of an activated NTA chip. The second flow cell is notactivated as a reference for non-specific interaction of the analyte tothe sensor chip. Various concentrations of target protein (1 nM-1 μMrange), serially diluted into the same assay buffer containing 1 μMcompound (1% DMSO final), are injected onto the FKBP12 surface andreference surface at a flow rate of 10 μl/min. The surface isregenerated between analyte injections with 350 mM EDTA.

Data Fitting: The BiaEvaluation software program is used for datafitting. All data is reference subtracted against both the referenceflow cell and a buffer injection. For kinetic analyses, data is locallyfit to a 1:1 interaction model.

Example 12. Determination of Binding of FKBP12/Compound 2 Complex toCEP250 by SPR

This protocol utilizes Surface Plasmon Resonance (SPR) as a method todetermine kinetics (K_(D), K_(a), K_(d)) for the binding of CEP250(analyte) to immobilized FKBP12-Compound 2 binary complex (ligand).

Reagents:

Compound 2 in 100% DMSO (in-house), 10×HBS-P+ buffer (GE HealthcareBR-1006-71), Assay buffer (1×HBS-P+ buffer, 1% DMSO, 1 μM Compound 2),12×HIS tagged FKBP12 (in-house), CEP25029.2 (residues 1982-2231) andCEP25011.4 (residues 2134-2231) (in-house).

Equipment:

BIACORE™ X100 (GE Healthcare)

Supplies:

NTA Sensor chip (GE Healthcare BR-1000-34)

Experimental Protocol:

Experiments are performed at 25° C. Stock solution of 12×HIS taggedFKBP12 is diluted to 100 nM in assay buffer containing 1 μM Compound 2(1% DMSO final). Approximately 200-400 RU of FKBP12 is immobilized onone of two flow cells of an activated NTA chip. The second flow cell isnot activated as a reference for non-specific interaction of the analyteto the sensor chip. Various concentrations of CEP250 (1 nM-1 μM range),serially diluted into the same assay buffer containing 1 μM Compound 2(1% DMSO final), are injected onto the FKBP12 surface and referencesurface at a flow rate of 10 μl/min. The surface is regenerated betweenanalyte injections with 350 mM EDTA.

Data Fitting:

The BiaEvaluation software program is used for data fitting. All data isreference subtracted against both the reference flow cell and a bufferinjection. For kinetic analyses, data is locally fit to a 1:1interaction model.

Results:

The k values for the binding of the FKBP12/Compound 2 complex toCEP25011.4 and CEP25029.2 are: K_(a) (1/Ms): 5.71×10⁵; K_(d) (1/s):3.09×10⁻³; and K_(D): 5.4 nM and K_(a) (1/Ms): 3.11×10⁵; K_(d) (1/s):9.25×10⁻⁵; and K_(D): 0.29 nM, respectively.

Example 13. Determination of Binding of Presenter Protein/CompoundComplexes to Target Proteins by ITC

General Protocol

This protocol utilizes Isothermal Titration Calorimetry (ITC) todirectly measure the heat change associated with binding of presenterprotein (e.g. FKBP, cyclophilin)-compound binary complexes to targetproteins. Measurement of the heat change allows accurate determinationof association constants (K_(a)), reaction stoichiometry (N), and thechange in binding enthalpy (ΔH).

Reagents:

Compounds in 100% DMSO (in-house), Protein Buffer (10 mM HEPES, pH 7.5,75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer+1% DMSO),presenter protein (e.g. FKBP, cyclophilin) (in-house), target protein(in-house).

Equipment:

MicroCal™ ITC₂₀₀ (GE Healthcare)

Experimental Protocol:

presenter protein (e.g. FKBP, cyclophilin) stock solution is diluted to10 μM in assay buffer (1% DMSO final). Compound is added to presenterprotein to 20 μM (1% DMSO final), and binary complex is filled into thereaction cell of the ITC device after 5-10 min pre-incubation time.Target protein stocks are diluted to 50 μM in assay buffer andsupplemented with 20 μM compound (1% DMSO final) before being filledinto the injection syringe. A control experiment in the absence ofcompound is also run to determine the heat associated with operationalartifacts and the dilution of titrant as it is injected from the syringeinto the reaction cell. Data collection and analysis are as describedfor binding of FKBP12-Compound 2 and FKBP12-Compound 6 binary complexesto CEP250.

Example 14. Determination of Binding of FKBP12/Compound 2 andFKBP12/Compound 11 Complexes to CEP250 by ITC

This protocol utilizes Isothermal Titration Calorimetry (ITC) todirectly measure the heat change associated with binding ofFKBP12-Compound 2 and FKBP12-Compound 6 binary complexes to CEP250.Measurement of the heat change allows accurate determination ofassociation constants (K_(a)), reaction stoichiometry (N), and thechange in binding enthalpy (ΔH).

Reagents:

Compound 2 and Compound 6 in 100% DMSO (in-house), Protein Buffer (10 mMHEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer+1%DMSO), FKBP12 (in-house), CEP25029.4 (residues 1982-2231) and CEP25011.4(residues 2134-2231) (in-house).

Equipment:

MicroCal™ ITC200 (GE Healthcare) Experimental Protocol: FKBP12 stocksolution was diluted to 10 μM in assay buffer (1% DMSO final). Compoundwas added to FKBP12 to 20 μM (1% DMSO final), and binary complex wasfilled into the reaction cell of the ITC device after 5-10 minpre-incubation time. CEP250 protein stocks were diluted to 50 μM inassay buffer and supplemented with 20 μM compound (1% DMSO final) beforebeing filled into the injection syringe. A control experiment in theabsence of compound was also run to determine the heat associated withoperational artifacts and the dilution of titrant as it was injectedfrom the syringe into the reaction cell. More detailed experimentalparameters are shown in Tables 10 and 11, below:

TABLE 10 ITC Experimental Parameters Experimental device: MicroCal ™iT₂₀₀(GE Healthcare) sample cell volume [μl] 270 injector volume [μl] 40Experimental parameters Total # of Injections 19 Cell Temperature [° C.]25 Reference Power [μCal/s] 5 Initial Delay [s] 200 Stirring Speed [rpm]750 Injection parameters Volume [μl] 2 Duration [s] 4 Spacing [s]170-200 Filter Period [s] 5 Feedback Mode/Gain high

TABLE 11 Protein and Ligand Concentrations for ITC Final protein andligand concentrations DMSO assay cell content syringe content ligandconc. [%] FKBP12, 10 μM CEP250_(29.4), 50 μM none 1.0 FKBP12, 10 μMCEP250_(11.4), 50 μM none 1.0 FKBP12, 10 μM CEP250_(29.4), 118 μMCompound 2, 20 μM 1.0 FKBP12, 10 μM CEP250_(29.4), 118 μM Compound 6, 20μM 1.0 FKBP12, 10 μM CEP250_(11.4), 68 μM Compound 2, 20 μM 1.0 FKBP12,10 μM CEP250_(11.4), 68 μM Compound 6, 20 μM 1.0

Data Fitting:

Data were fitted with the Origin ITC200 software according to thefollowing procedure:

-   -   1) Read raw data    -   2) In “mRawITC”: adjust integration peaks and baseline,        integrate all peaks    -   3) In “Delta H”—data control: remove bad data (injection #1 and        other artifacts), subtract straight line (background        subtraction)    -   4) In “Delta H”—model fitting: select one set of sites model,        perform fitting with Levenberg-Marquardt algorithm until Chi        Square is not reduced further, finish with “done” (parameters N,        K_(a) and ΔH are calculated based on fitting) ITC measurements        for the binding of FKBP12-Compound 2 and FKBP12-Compound 6        binary complexes to CEP250 are summarized in Table 12 below.

TABLE 12 ITC Measurements ΔH −T*ΔS ΔG cell syringe T Kd [kJ*mol-[kJ*mol- [kJ*mol- Experiment content content ligand [K] N [μM]* mol−1]**mol−1]*** mol−1]**** 3 FKBP12, CEP25029.4, none 298 ND ND ND ND ND 10 μM118 μM 4 FKBP12, CEP25011.4, none 298 ND ND ND ND ND 10 μM 68 μM 5FKBP12, CEP25029.4, Compound 298 0.50 0.19 −52.21 13.80 −38.41 10 μM 118μM 2, 20 μM 6 FKBP12, CEP25029.4, Compound 298 0.57 0.36 −58.48 21.73−36.74 10 μM 118 μM 6, 20 μM 7 FKBP12, CEP25011.4, Compound 298 0.560.07 −49.37 8.62 −40.75 10 μM 68 μM 2, 20 μM 8 FKBP12, CEP25011.4,Compound 298 0.54 0.08 −47.78 7.41 −40.36 10 μM 68 μM 6, 20 μM*K_(d)(calculated from K_(a) = 1/K_(d)) **ΔH ***T*ΔS (calculated fromequation (−TΔS = ΔG − ΔH) ****ΔG = −RT In K_(a) = RT In K_(d)

Results:

Overall, the data for FKBP12-Compound 2 and FKBP12-Compound 6 binarycomplexes binding to CEP25011.4 and CEP25029.4 showed similarinteraction parameters. K_(d) values were similar for all combinations.All interactions showed an almost identical thermodynamic profile inwhich binding is characterized by a purely enthalpic binding mode (−T*ASterm is positive and does not contribute to the Gibbs free energy).Binding stoichiometries for all interactions were N═0.5-0.6 and supporta 1:2 binding ratio for 1 CEP250 homodimer binding to 2 FKBP12molecules, as evidenced in the crystal structure ofCEP25011.4/F2/FKBP12.

Example 15. Crystallographic Structural Determination of TertiaryComplexes

General Protocol

This protocol describes the crystallization and structure determinationmethod for structures of specific FKBP12-compound-target protein ternarycomplexes.

Reagents:

Compound in 100% DMSO (in-house), FKBP12 (in-house), and mammaliantarget protein (in-house).

Equipment:

Superdex 200 (GE Healthcare)

Experimental Protocol:

A 3:1 molar excess of compound is added to FKBP12 in 12.5 mM HEPES pH7.4, 75 mM NaCl buffer, and incubated overnight at 4° C. A 3:1 molarexcess of FKBP12-compound binary complex is added to target protein andincubated at 4° C. overnight to complete ternary complex formation. Pureternary complex is isolated by gel filtration purification on a Superdex200 column in 12.5 mM HEPES pH 7.4, 75 mM NaCl. Purified complex (at10-20 mg/ml) is subjected to crystallization at 22° C. using sittingdrop vapor diffusion using various buffers, surfactants and saltsolutions. For data collection, crystals are transferred to a solutioncontaining mother liquor supplemented with 20-25% glycerol, and thenfrozen in liquid nitrogen. Diffraction datasets are collected at theAdvanced Photon Source (APS) and processed with the HKL program.Molecular replacement solutions are obtained using the program PHASER inthe CCP4 suite, using the published structure of FKBP12 (PDB-ID 1 FKD)as a search model. Subsequent model building and refinement areperformed according to standard protocols, e.g., with the softwarepackages CCP4 and COOT.

Example 16. Crystallographic Structural Determination of TertiaryComplexes of FKBP12/Compound 2 and FKBP12/Compound 6 Complexes withCEP250

This protocol describes the crystallization and structure determinationmethod for structures of FKBP12-Compound 2-CEP250 and FKBP12-Compound6-CEP250 ternary complexes.

Reagents:

Compound 2 and Compound 6 in 100% DMSO (in-house), FKBP12 (in-house),and CEP25011.4 (residues 2134-2231) (in-house).

Equipment:

Superdex 200 (GE Healthcare)

Experimental Protocol:

A 3:1 molar excess of Compound 2 or Compound 6 was added to FKBP12 in12.5 mM HEPES pH 7.4, 75 mM NaCl buffer, and incubated overnight at 4°C. A 3:1 molar excess of FKBP12-Compound 2 or FKBP12-Compound 6 binarycomplex was added to CEP25011.4 and incubated at 4° C. overnight tocomplete ternary complex formation. Pure ternary complex was isolated bygel filtration purification on a Superdex 200 column in 12.5 mM HEPES pH7.4, 75 mM NaCl. Purified complex (at 10-20 mg/ml) was subjected tocrystallization at 22° C. using sitting drop vapor diffusion.FKBP12-Compound 2-CEP250 crystals were grown in a well solutioncontaining 0.2 M sodium malonate, 0.1 M HEPES 7.0, 21% PEG 3350.FKBP12-Compound 6-CEP250 crystals were grown in a well solutioncontaining 0.1 M Tris pH 8.5, 0.2 M trimethylamine N-oxide, 22-24%PEG2000 MME. For data collection crystals were transferred to a solutioncontaining mother liquor supplemented with 20-25% glycerol, and thenfrozen in liquid nitrogen. Diffraction datasets were collected at theAdvanced Photon Source (APS) and processed with the HKL program.Molecular replacement solutions were obtained using the program PHASERin the CCP4 suite, using the published structure of FKBP12 (PDB-ID 1FKD) as a search model. Subsequent model building and refinement wereperformed according to standard protocols with the software packagesCCP4 and COOT.

Results:

Overall structure of FKBP12-Compound 2-CEP250: In the structure ofFKBP12 with CEP250 in complex with Compound 2, two FKBP12 monomers werebound to a homodimer of CEP250. The two CEP250 monomers formed acoiled-coil structure. There were four hetero dimers on the asymmetricunit with basically the same overall conformation. The model comprisedresidues Met1 to Glu108 of FKBP12 and Asp2142 to His2228 of CEP250. Theelectron density showed an unambiguous binding mode for the ligandCompound 2, including the orientation and conformation of the ligand.

The CEP250 residues involved in binding Compound 2 are L2190, Q2191,V2193, A2194, M2195, F2196, L2197, and Q2198. The CEP250 residuesinvolved in binding to FKBP12 are A2185, S2186, S2189, Q2191, M2195,Q2198, V2201, L2202, R2204, D2205, S2206, Q2208, Q2209, and Q2212.

The total buried surface area of the ternary complex is 1759 Å². Thetotal buried surface area of CEP250 is 865 Å² of which 663 Å² iscontributed by FKBP12 and 232 Å² is contributed by Compound 2. 100% ofthe binding interactions in the ternary complex between Compound 2 andCEP250 were van der Waals or pi-pi interactions. By comparison, 100% ofthe binding interactions between rapamycin and mTOR are van der Waals orpi-pi interactions, and 89% of the binding interactions between FK506and calcineurin are van der Waals or pi-pi interactions while 11% arehydrogen bonds (two H-bonds from C13 and C15 OMe to Trp 352 N—H).

Overall structure of FKBP12-F11-CEP250: In the structure of FKBP12 withCEP250 in complex with F11, one FKBP12 monomer was bound to a homodimerof CEP250. The two CEP250 monomers formed a coiled-coil structure. Thecrystals contain a heterotrimer (one FKBP12 and two CEP250) in theasymmetric unit. The model comprised residues Met1 to Glu108 of FKBP12and Ser2143 to His2228 of CEP250. One short loop region of FKBP12(18-19) was not fully defined by electron density and was not includedin the model. The electron density showed an unambiguous binding modefor the ligand F11, including the orientation and conformation of theligand.

The CEP250 residues involved in binding F2 are L2190, Q2191, V2193,A2194, M2195, F2196, L2197, and Q2198. The CEP250 residues involved inbinding to FKBP12 are Q2182, A2185, S2186, S2189, Q2191, M2195, Q2198,V2201, L2202, R2204, D2205, S2206, Q2208, Q2209, and Q2212.

The total buried surface area of the ternary complex is 1648 Å². Thetotal buried surface area of CEP250 is 831 Å² of which 590 Å² iscontributed by FKBP12 and 241 Å² is contributed by F2.

Statistics of the final structures are listed in Table 13 and 14 below.

TABLE 13 FKBP12-Compound 2-CEP250 Ligand Compound 2 Resolution [Å]136.05-2.20 Number of reflections (working/test) 50564/2723 R_(cryst)[%] 20.8 R_(free)[%]² 25.6 Total number of atoms: Protein 6146 Water 226Ligan 176 PEG 273 Magnesium 1 Maltose 7 Deviation from ideal geometry: ³Bond lengths [Å] 0.007 Bond angles [°] 1.17 Bonded B's [Å²]⁴ 5.4Ramachandran plot: ⁵ Most favoured regions [%] 94.6 Additional allowedregions [%] 4.8 Generously allowed regions [%] 0.6 Disallowed region [%]0.0 ¹Values as defined in REFMAC5, without sigma cut-off ²Test-setcontains 2.4% of measured reflections ³ Root mean square deviations fromgeometric target values ⁴Calculated with MOLEMAN ⁵ Calculated withPROCHECK

TABLE 14 FKBP12-Compound 6-CEP250 Ligand Compound 6 Resolution [Å]72.84-2.10 Number of reflections (working/test) 16398/877 R_(cryst) [%]24.2 R_(free)[%]² 29.9 Total number of atoms: Protein 2224 Water 31Ligand 44 PEG 14 Deviation from ideal geometry: ³ Bond lengths [Å] 0.008Bond angles [°] 1.07 Bonded B's [Å²]⁴ 3.0 Ramachandran plot: ⁵ Mostfavoured regions [%] 93.5 Additional allowed regions [%] 4.9 Generouslyallowed regions [% ] 1.6 Disallowed region [%] 0.0 ¹Values as defined inREFMAC5, without sigma cut-off ²Test-set contains 5.1% of measuredreflections ³ Root mean square deviations from geometric target values⁴Calculated with MOLEMAN ⁵ Calculated with PROCHECK

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A macrocyclic compound, or a pharmaceutically acceptable saltthereof, having the structure:

wherein A is a target protein interacting moiety described by thestructure of Formula XIII:

wherein the dotted lines represent zero to three double bonds, providedthat no two double bonds are adjacent to one another; R³¹ and R³² areindependently hydrogen, hydroxyl, optionally substituted amino, halogen,thiol, optionally substituted amino acid, optionally substituted C₁-C₆acyl, optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substitutedC₁-C₆ heteroalkyl, optionally substituted C₂-C₆ heteroalkenyl,optionally substituted C₂-C₆ heteroalkynyl, optionally substitutedC₃-C₁₀ cycloalkyl, optionally substituted C₄-C₁₀ cycloalkenyl,optionally substituted C₄-C₁₀ cycloalkynyl, optionally substitutedC₅-C₁₀ aryl, optionally substituted C₅-C₁₀ aryl C₁-C₆ alkyl, optionallysubstituted C₂-C₉ heteroaryl, optionally substituted C₂-C₉ heteroarylC₁-C₆ alkyl, optionally substituted C₂-C₉ heterocyclyl, optionallysubstituted C₂-C₉ heterocyclyl C₁-C₆ alkyl, or R³¹ and R³² combine toform C═O; R³³ is hydrogen or C═O, provided that no double bond isadjacent to a C═O; and B is a presenter protein binding moiety describedby a structure selected from:

and each of L¹ and L² is, independently, a linker.
 2. The compound ofclaim 1, or a pharmaceutically acceptable salt thereof, wherein thetarget protein interacting moiety is described by a structure selectedfrom:


3. The compound of claim 2, or a pharmaceutically acceptable saltthereof, wherein the target protein interacting moiety is described by astructure selected from:


4. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein the presenter protein binding moiety is described by astructure selected from:


5. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein L¹ and L² are each, independently, selected from a bondor a linear chain of up to 10 atoms, independently selected from carbon,nitrogen, oxygen, sulfur and phosphorous atoms, wherein each atom in thechain is optionally substituted with one or more substituentsindependently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl,chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino,alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio,alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate,phosphoryl, and sulfonyl, and wherein any two atoms in the chain may betaken together with the substituents bound thereto to form a ring,wherein the ring may be further substituted and/or fused to one or moreoptionally substituted carbocyclic, heterocyclic, aryl, or heteroarylrings.
 6. The compound of claim 5, or a pharmaceutically acceptable saltthereof, wherein L¹ and L² are each a bond.
 7. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein: the targetprotein interacting moiety is described by:

the presenter protein binding moiety is described by:

and L¹ and L² are each a bond.
 8. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein: the target proteininteracting moiety is described by:

the presenter protein binding moiety is described by:

and L¹ and L² are each a bond.
 9. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein: the target proteininteracting moiety is described by:

the presenter protein binding moiety is described by:

and L¹ and L² are each a bond.
 10. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein: the target proteininteracting moiety is described by:

wherein R³¹ is hydrogen, and R³² is hydroxyl; the presenter proteinbinding moiety is described by:

and L¹ and L² are each a bond.
 11. A pharmaceutical compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient.
 12. A presenterprotein/compound complex comprising a compound of claim 1, or apharmaceutically acceptable salt thereof, and a presenter protein.
 13. Atripartite complex including (i) the presenter protein/compound complexof claim 12; and (ii) a target protein.
 14. The tripartite complex ofclaim 13, wherein the presenter protein is a prolyl isomerase.
 15. Thetripartite complex of claim 14, wherein the target protein is CEP250.16. A method of modulating a target protein comprising contacting a cellexpressing said target protein and a presenter protein with an effectiveamount of a compound of claim 1, or a pharmaceutically acceptable saltthereof, under conditions wherein the compound can form a complex withthe presenter protein and the resulting complex can bind to said targetprotein, thereby modulating said target protein.
 17. The method of claim16, wherein the presenter protein is a prolyl isomerase.
 18. The methodof claim 17, wherein the target protein is CEP250.
 19. A method for thepreparation of a compound of claim 1 comprising culturing a bacterialstrain of the genus Streptomyces and isolating the compound from thefermentation broth.
 20. A method of treating an infection comprisingadministering an effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof, to a subject in need thereof.